Genes encoding desulfurization enzymes

ABSTRACT

This invention provides novel genes encoding enzymes which decompose difficult-to-decompose thiophene compounds. By using these genes, sulfur atoms can be released from the thiophene compounds in fossil fuel such as petroleum, and the diffusion of sulfur into the environment caused by the combustion of the fossil fuel can be prevented.

This is a divisional application of U.S. patent application Ser. No.09/647,540, filed Sep. 29, 2000, now U.S. Pat. No. 6,420,158 which wasthe National Stage of International Application No. PCT/JP99/01756,filed Apr. 2, 1999, which in turn claims the benefit of JapaneseApplication No. 090387/1998, filed Apr. 2, 1998, and JapaneseApplication No. 310545/1998, filed Oct. 30, 1998.

TECHNICAL FIELD

The present invention relates to enzymes having the function ofdecomposing, using microorganisms, thiophene compounds, namelybenzothiophene, dibenzothiophene (hereinafter referred to as “DBT”) andtheir substituted compounds, or derivatives thereof, and genes encodingthe enzymes. By using the enzymes and the gene defined in the presentinvention, sulfur can be released from benzothiophene, DBT and theirsubstituted compounds, or derivatives thereof which are contained infossil fuels such as petroleum. As a result, sulfur, which is generallydiffused in the air when fossil fuels such as petroleum and coal areburned, can be easily removed from the fossil fuel.

PRIOR ART

In order to remove sulfur from hydrocarbon fuel such as petroleum,methods including alkali treating or solvent desulfurization are known.However, at present, mainly hydrodesulfurization is used.Hydrodesulfurization is a method for reacting sulfur compounds in apetroleum fraction with hydrogen in the presence of a catalyst andremoving the produced hydrogen sulfide so as to obtain low-sulfurproducts. As a catalyst, metallic catalysts such as cobalt, molybdenum,nickel and tungsten are used with alumina as a carrier. When themolybdenum on alumina is used as the catalyst, usually cobalt or nickelis added as a promoter to enhance catalysis performance. Thehydrodesulfurization with metallic catalysts is undoubtedly a fineprocess which is widely used throughout the world at the moment.However, as a process for producing petroleum products adapted to morestrict environmental regulations, there are some problems. Some examplesare discussed below briefly.

Generally the substrate specificity of a metallic catalyst is low, andso it is suitable for decomposing various kinds of sulfur compounds andlowering the amount of sulfur contained in the fossil fuel as a whole.However, it is considered that the effect of desulfurization withmetallic catalyst is sometimes insufficient for a specific group ofsulfur compounds, i.e., heterocyclic sulfur compounds such asbenzothiophene, DBT and their alkyl derivatives. For example, afterdesulfurizing light oil, various heterocyclic organic sulfur compoundsstill remain. One reason why the effect of desulfurization with metalliccatalyst is insufficient would be steric hindrance caused bysubstituents which are around the sulfur atoms of the organic sulfurcompounds. Among these substituted compounds, the influence of a methylsubstituted compound on the reaction of a metallic catalyst has beenstudied in relation to thiophene, benzothiophene, DBT and so on.According to such studies, it is generally said that, as the number ofsubstituted compounds increases, desulfurization reaction ratesdecreases. However, it is also said that the position of thesubstituents have a very large influence on the reactivity. One of thereports which have shown that the steric hindrance has the significantinfluence on the reaction of metallic catalyst is, for example, Houalla,M., Broderick, D. H., Sapre, A. V., Nag, N. K., de Beer, V. H., Gates,B. C., Kwart, H. J., Catalt., 61, 523-527(1980). In fact, it is knownthat a considerable amount of various alkyl derivatives of DBT exists inlight oil (e.g. Kabe, T., Ishihara, A. and Tajima, H. Ind. Eng. Chem.Res., 31, 1577-1580(1992)).

As stated above, it is considered that, in order to desulfurize organicsulfur compounds which are resistant against hydrodesulfurization,higher reaction temperature and pressure than that usually used arerequired, and also the amount of hydrogen added to be increasedremarkably. It is thus expected that enormous capital investment andoperating costs are needed to improve a hydrodesulfurization processsuch as this. For example, light oil contains organic sulfur compoundsresisting such hydrodesulfurization as a major compound species, and asstated above, a substantial improvement on the hydrodesulfurizationprocess is required to carry out more sophisticated desulfurization oflight oil (an ultra deep desulfurization).

On the other hand, the enzyme-reaction in an organism proceeds underrelatively mild conditions, and further, the rate of enzyme reaction inan organism compares favorably with that of a chemical catalyst.Moreover, there are so many kinds of enzymes in vivo to conformappropriately to various kinds of vital reactions occurring therein, andthose enzymes usually show a very high substrate specificity. Thesecharacteristics are expected to be utilized for so-calledbiodesulfurization reaction, which removes sulfur from sulfur compoundsin fossil fuel by using microorganisms (Monticello, D. J., HydrocarbonProcessing 39-45(1994)).

There are a large number of reports on methods for removing sulfur fromheterocyclic sulfur compounds which are ingredients of petroleum byusing bacteria, and these methods are broadly divided into the reactionof decomposing a ring (C—C bond cleavage) and the C—S bond cleavagereaction. As bacteria having C—C-bond—attacking desulfurizationactivity, for example, strains belonging to Pseudomonas sp., Pseudomonasaeruginosa, Beijerinckia sp., Pseudomonas alcaligenes, Pseudomonasstutzeri, Pseudomonas putida, Brevibacterium sp. are known. Thesebacteria carry out the cleavage of C—C bond in heterocyclic sulfurcompounds of which a representative example is DBT, decompose a benzenering, thereafter, by oxidative reaction cascade, they conduct ametabolism in which salt containing sulfur atom(s) is released. As thereaction mechanism of the carbon-backbone-attacking pathway, there arethe hydroxylation of aromatic ring (DBT→→1,2-dihydroxy DBT), thecleavage of a ring, and the oxidation to water-soluble product(1,2-dihydroxy DBT→→trans-4 [2-(3-hydroxy)thianaphthenyl]-2-oxo-butenoic acid, 3-hydroxy-2-formylbenzothiophene),and this reaction mechanism is called “Kodama pathway”. The C—C bond ina benzene ring of DBT is attacked by this kind of reaction to generatevarious water-soluble substances which are extractable from the oil. Dueto this reaction, however, other aromatic molecules in the oil are alsoattacked, and as a result, a significant amount of hydrocarbons move towater phase (Hartdegen, F. J., Coburn, J. M. and Roberts, R. L. Chem.Eng. Progress, 80, 63-67(1984)). This causes the reduction of totalcalories of petroleum and so it is an industrially ineffective reaction.Furthermore, as Kodama et al. has reported, this type of bacteriaoxidatively decomposing DBT provides water-soluble thiophene compounds(mainly 3-hydroxy-2-formylbensothiophene) as oxidized products, but thisis a substance difficult to remove from water phase. In addition, sincethe attack to the carbon ring of DBT often occurs at position 2 or 3 ofDBT, DBT substituted with an alkyl or alkyl groups at these positionsdoes not become the substrate of the Kodama pathway.

It has been reported that there are microorganisms which decompose notonly crude oil or coal but also model compounds containing sulfur,remove selectively hetero-atom sulfur, and generate sulfate and hydroxylcompounds. Taking the structure of the metabolites into consideration,this kind of reaction is considered to be one which cleaves specificallyC—S bond in sulfur compounds and accordingly releases sulfur in the formof sulfate. As shown in Table 1, to date, some biodesulfurizationreaction systems which are characterized by attacking sulfur have beenreported.

TABLE 1 C—S bond attacking bacteria DECOMPOSED REFERENCE STRAINSUBSTRATE PRODUCT DOCUMENTS Pseudomonas sp. dibenzothiophene; hydroxybi-Isbister et al. CB1 coal phenyl + sulfate (1985) Acinetobacter sp.dibenzothiophene hydroxybi- Isbister et al. CB2 phenyl + sulfate (1985)Gram-positive coal sulfate Crwaford et al. bacteria (1990) Rhodococcusdibenzothiophene hydroxybi- Kilbane (1989) rhodochrous coal; petroleumphenyl + sulfate IGTS8 (ATCC 53968) Desulfovibrio dibenzothiophenebiphenyl + Kim et al. desulfuricans hydrogen sulfide (1990)Corynebacterium dibenzothiophene hydroxybi- Omori et at. sp. phenyl +sulfate (1992) Brevibacterium dibenzothiophene benzoic acid + vanAfferden sp. DO sulfite et al. (1990) Gram-positive dibenzothiophenebiphenyl + Finnerty (1993) bacterium FE-9 hydrogen sulfide thianthrenebenzene + hydrogen sulfide Pseudomonas sp. benzilmetyl- benzaldehyde vanAfferden OS1 sulfide et al. (1990) Rhodococcus dibenzothiophenehydroxybiphenyl Wang et al. erythropolis (1994) Rhodococcusdibenzothiophene hydroxybiphenyl Izumi et al. erythropolis (1994)., D-1,H-2 Ohshiro et al. (1995) Agrobacterium dibenzothiophene hydroxybiphenylConstantl et al. sp. (1994) Xanthomonas sp. dibenzothiophenehydroxybiphenyl Constantl et al. (1994) Arthrobacter K3b dibenzothio-benzoic acid + Dahlberg phenesulfone sulfite (1992)

For all biodesulfurizations stated above, a metabolic reaction ofmicroorganism cultured at around 30° C. is used. On the other hand, itis known that generally the rate of chemical reaction increases as thetemperature becomes higher. Regarding the desulfurization in petroleumrefining process, fractional distillation or desulfurization reaction iscarried out under conditions of high temperature and high pressure.Therefore, when biodesulfurization is incorporated into the petroleumrefining process, it is desirable that the desulfurization reaction iscarried out at higher temperature in the mid course of cooling process,without cooling the fraction to room temperature. Some reports onhigh-temperature biodesulfurization are as follows.

Most attempts to carry out the desulfurization reaction usingmicroorganisms at room temperature are directed to coal desulfurization.Coal contains various kinds of sulfur compounds. The main inorganicsulfur compound is pyrite. On the other hand, the organic sulfurcompounds vary widely in type, and it is known that the majority ofthese contain thiol, sulfide, disulfide and thiophene groups. Themicroorganisms used are Sulfolobus bacteria which are all thermophiles.There are several reports that various Sulfolobus strains were used inthe leaching of metal out of mineral sulfide (Brierley C. L. & Murr, L.E., Science 179, 448-490(1973)), the desulfurization of pyrite in coal(Kargi, F. & Robinson, J. M., Biotechnol. Bioeng, 24, 2115-2121(1982);Kargi, F. & Robinson, J. M., Appl. Environ. Microbiol., 44,878-883(1982); Kargi, F. & Cervoni, T. D., Biotechnol. Letters 5,33-38(1983); Kargi, F. and Robinson, J. M., Biotechnol. Bioeng., 26,687-690(1984); Kargi, F. & Robinson, J. M., Biotechnol. Bioeng. 27,41-49(1985); Kargi, F., Biotechnol. Lett., 9, 478-482(1987)) and so on.According to Kargi and Robinson (Kargi, F and Robinson, J. M., Appl.Environ. Microbiol., 44, 878-883(1982)), a certain strain of Sulfolobusacidocaldarius isolated from an acidic thermal spring of YellowstoneNational Park in U.S.A. grows at 45-70° C. and oxidizes elemental sulfurat an optimum pH2. Furthermore, it has been also reported that two otherkinds of Sulfolobus acidocaldarius stains oxidize pyrite (Tobita, M.,Yokozeki, M., Nishikawa, N. & Kawakami, Y., Biosci. Biotech. Biochem.58, 771-772(1994)).

It is known that, among the organic sulfur compounds contained in fossilfuel, DBT and its substituted compounds, or derivatives thereof, aregenerally resistant to hydrodesulfurization in the petroleum refiningprocess. High-temperature decomposition by Sulfolobus acidocaldaius(hereinafter, referred to as “S. acidocaldarius”) of the said DBT hasbeen also reported (Kargi, & Robinson, J. M., Biotechnol. Bioeng, 26,687-690(1984); Kargi, F., Biotechnol. Letters 9, 478-482(1987)).

According to these reports, when model aromatic heterocyclic sulfurcompounds such as thianthrene, thioxanthene, DBT and the like arereacted with S. acidocaldarius at high temperature, these sulfurcompounds are oxidized and decomposed. Oxidation of these aromaticheterocyclic sulfur compounds by this microorganism is observed at 70°C. and it results in the formation of sulfate ions as the reactionproduct. However, because this reaction is carried out in a medium whichdoes not contain any carbon source other than sulfur compounds, thesesulfur compounds would be also used as the carbon sources. That is tosay, it is clear that C—C bond in sulfur compounds was decomposed.Furthermore, S. acidocaldarius can be grown only in an acidic medium,and the oxidative decomposition reaction require under severely acidicconditions (e.g. pH2.5) to continue. Since such conditions cause thedegradation of petroleum products and at the same time requiresacid-resistant materials in the desulfurization-associated step, it isconsidered not to be desirable for the process. When S. acidocaldariusis grown under autotrophic conditions, the microorganism acquiresnecessary energy from reduced iron-sulfur compounds and uses carbondioxide as the carbon source. Alternatively, when S. acidocaldarius isgrown under heterotrophic conditions, it can use various organiccompounds as carbon and energy sources. In other words, it can be saidwhen fossil fuel exists, it can be used as a carbon source.

Finnerty et al. has reported that the strains belonging to Pseudomonasstutzeri, Pseudomonas alcaligenes and Pseudomonas putida decompose DBT,benzothiophene, thioxanthene and thianthrene, and convert them intowater-soluble substances (Finnerty, W. R., Shockiey, K., Attaway, H. inMicrobial Enhanced Oil Recovery, Zajic, J. E. et al.(eds.) PenwellTuisa, Okia, 83-91(1983)). In this case, the oxidative reaction canproceed at 55° C. However, the decomposed products of DBT by thesePseudomonas strains are 3-hydroxy-2-formylbenzothiophene reported byKodama et al. (Monticello, D. J., Bakker, D., Finnerty, W. R. Appl.Environ. Microbiol., 49, 756-760(1985)). The oxidation activity of DBTby the Pseudomonas strains is induced by an aromatic hydrocarbon withoutsulfur such as naphthalene or salicylic acid, and is blocked bychloramphenicol. From this fact, it was found that the decompositionreaction of DBT by the Pseudomonas strains is based on the cleavage of aC—C bond in aromatic ring. Moreover, there is the risk that valuablearomatic hydrocarbons other than sulfur compounds in the petroleumfraction are also decomposed together with them, and if this occurs, itresults in lowering of fuel value or petroleum fraction quality.

As stated above, the known strains which can decompose DBT at hightemperature are the ones which catalyze the reaction of cleaving a C—Cbond in the DBT molecule and use the resulting compounds as carbonsources. As mentioned above, the decomposition reaction of organicsulfur compounds which cleaves specifically C—S bond but leaves C—C bondunchangeable is desirable as a real method for desulfurizing petroleum.In other words, the most desirable biodesulfurization process is onewhich has an activity of cleaving C—S bond in the molecule of DBT andits alkyl-substituted compounds, or their derivatives at hightemperature and uses microorganisms which generate desulfurizationproducts in the form of water-soluble substances.

As stated above, several families of bacteria are known asmicroorganisms conducting the C—S bond cleavage to decompose DBT.However, of all these bacteria, there were found no examples describedto have an activity of decomposing DBT under high temperature conditionsof more than 42° C. For example, ATCC53968 (Rhodococcus sp). is athoroughly studied DBT-decomposing strain and conducts an addition of anoxygen atom to the sulfur atom of DBT, generating DBT sulfone(hereinafter referred to as “DBTO2”) from DBT sulfoxide (hereinafterreferred to as “DBTO”), and further generating 2-hydroxybiphenyl(hereinafter referred to as “2-HBP”) via 2-(2′-hydroxyphenyl)benzensulfinate. However, it has been reported that even this straingrows very slowly or stops growing, when it is cultured for 48 hours ata temperature of 37° C. or 43° C. which is slightly higher than 30° C.(an ordinary culturing temperature) (Japanese Patent ApplicationLaying-Open (kokai) No. 6-54695). Therefore, it has been presumed thatthe use of the microorganism, which can grow under high temperaturescondition and can cleave specifically the C—S bond of heterocyclicsulfur compounds including organic sulfur compounds, especially DBT, itssubstituted compounds, or their derivatives at high temperature, is moresuitable for conducting the desulfurization reaction at hightemperature. The present inventors have conducted a wide range ofscreenings, has amplified the microorganisms under high temperatureconditions, nearly 60° C., and has already isolated 2 strains ofPaenibacillus sp., which are high-temperature desulfurizing strainshaving a function of decomposing and desulfurizing DBT families for thefirst time in the world (Japanese Patent Application Laying-Open (kokai)No. 10-036859). If genes which are associated with high-temperaturedesulfurization activity can be isolated from this strain, it ispossible to endow a wide range of microbes with the function ofhigh-temperature desulfurization by using genetic engineering such asrecombinant DNA technology.

Among the bacteria known for their function of conducting C—S bondcleavages in the decomposition reaction, genes thereof, which encodeenzyme activities involved in DBT decomposition reaction that areidentified and whose nucleotide sequences are determined are, to thebest of the present inventors' knowledge, only dsz genes of Rhodococcussp. IGTS8 strain (Denome, S., Oldfleld., C., Nash, L. J. and Young, K.D. J.Bacteriol., 176:6707-6716, 1994; Piddington, C. S., Kovacevich, B.R. and Rambosek, J. Appl. Environ. Microbiol., 61:468-475, 1995). TheDBT decomposition reaction by IGTS8 strain is catalyzed by threeenzymes: DszC catalyzing the conversion from DBT to DBTO2 via DBTO, DszAcatalyzing the conversion from DBTO2 to 2-(2′-hydroxyphenyl)benzensulfinic acid, and DszB catalyzing the conversion from2-(2′-hydroxyphenyl) benzensulfinic acid to 2-HBP (Denome, S.,Oldfield., C., Nash, L. J. and Young, K. D. J.Bacteriol., 176:6707-6716,1994; Gray, K. A., Pogrebinshy, O. S., Mrachko, G. T., Xi, L.Monticello, D. J. and Squires, C. H. Nat Biotechnol., 14:1705-1709,1996; Oldfield, C., Pogrebinsky, O., Simmonds, J., Olson, E. S. andKulpa, C. F., Microbiology, 143:2961-2973, 1997). The genescorresponding to the above enzymes are called dszA, dszB and dszC. It isknown that the enzymes DszC and DszA are monooxygenases, and bothenzymes need the coexistence of NADH-FMN oxidoreductase activity fortheir oxygenation reaction (Gray, K. A., Pogrebinsky, O. S., Mrachko, G.T., Xi, L. Monticello, D. J. and Squires, C. H. Nat Biotechnol.,14:1705-1709, 1996; Xi, L. Squires, C. H., Monticello, D. J. and Childs,J. D. Biochem. Biophys. Res Commun., 230:73-76, 1997) It has beenreported that when the dsz genes are induced and expressed inEscherichia coli by shifting the temperature, DszA activity by cellculture reaches the maximum at 39° C., but remarkably decreases at 42°C. (Denome, S., Oldfield., D., Nash, L. J. and Young, K. D. J.Bacteriol., 176:6707-6716, 1994). This report corresponds to the resultof an experiment on resting cell reaction system which shows that thedesulfurization enzyme activity of IGTS8 strain reaches the maximumaround room temperature, but activity decreases at higher temperatureand there is no desulfurization activity at temperatures of more than50° C. (Konishi, J., Ishii, Y., Onaka, T., Okumura, K. and Suzuki, M.Appl. Environ. Microbiol., 63:3164-3169, 1997). Therefore, the geneswhich direct DBT-decomposing activity specific for C—S bond under hightemperature conditions, more than 50° C., have not been previouslyreported.

Objects to be Achieved by the Invention

One object of the present invention is to isolate the genes involved inhigh-temperature desulfurization reaction from a microorganism having anability of acting on benzothiophene and DBT compounds and decomposingthem at high temperature, to specify the structure (especially thenucleotide sequences), and to create novel desulfurizing microorganismsby introducing the genes into a heterologous microorganism and endowingit with the ability of desulfurization. Another object of the presentinvention is to establish a method for removing sulfur by actuallycontacting such a microorganism with benzothiophene, DBT and their alkylderivatives and cleaving the C—S bonds of these compounds.

Means to Achieve the Objects

After thorough studies directed to achieve the above objects, thepresent inventors have succeeded in isolating the genes involved indesulfurization reaction from high-temperature desulfurization bacteria,Paenibacillus sp., and have completed the present invention.

That is to say, the first aspect of the present invention relates togenes encoding desulfurization enzymes.

The second aspect of the present invention relates to vectors containingthe said genes.

The third aspect of the present invention relates to transformantscontaining the said vectors.

The forth aspect of the present invention relates to desulfurizationenzymes.

The fifth aspect of the present invention relates to genes encodingtransposase.

The sixth aspect of the present invention relates to transposase.

This specification includes part or all of the contents as disclosed inthe specifications and/or drawings of Japanese Patent Application Nos.10-090387 and 10-310545 which are priority documents of the presentapplication.

DISCLOSURE OF THE INVENTION

The details of the present invention are disclosed below.

(1) Gene Encoding a Desulfurization Enzyme

The genes of the present invention comprise the following three types ofgenes.

The first gene encodes (a) a protein represented by an amino acidsequence shown in SEQ ID NO: 2; or (b) a protein comprising a deletion,substitution or addition of one or more amino acids in the amino acidsequence of SEQ ID NO: 2, and having a function of converting DBTO2 into2-(2′-hydroxyphenyl) benzenesulfinic acid.

The second gene encodes (a) a protein represented by an amino acidsequence shown in SEQ ID NO: 4; or (b) a protein comprising a deletion,substitution or addition of one or more amino acids in the amino acidsequence of SEQ ID NO: 4, and having a function of converting2-(2′-hydroxyphenyl) benzenesulfinic acid into 2-HBP.

The third gene encodes (a) a protein represented by an amino acidsequence shown in SEQ ID NO: 6; or (b) a protein comprising a deletion,substitution or addition of one or more amino acids in the amino acidsequence of SEQ ID NO: 6, and having a function of converting DBT intoDBTO2 via DBTO.

The above-described first, second and third genes have a certainhomology to dszA, dszB or dszC derived from Rhodococcus sp. IGTS8strain. However, the proteins encoded by these genes are different fromthe ones encoded by dszA, dszB and dszC in terms of their properties.

Among the genes of the present invention, the ones which encode aminoacid sequences as shown in SEQ ID NOS: 2, 4 and 6 can be obtained by themethods described later in Examples. Since the nucleotide sequences ofthese genes have been already determined as shown in SEQ ID NOS: 1, 3and 5, they can also be obtained by synthesizing primers on the basis ofthese nucleotide sequences, and carrying out PCR using the primers and aDNA as a template, the DNA being prepared from Paenibacillus sp. A11-1strain (which was deposited with the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology underaccession No. FERM BP-6025 on Jul. 22, 1997) or A11-2 strain (which wasdeposited with the same international depositary authority underaccession No. FERM BP-6026 on Jul. 22, 1997).

The genes encoding amino acid sequences comprising a deletion,substitution or addition of one or more amino acids in the amino acidsequence of SEQ ID NOS: 2, 4 and 6 can be obtained by modifying thegenes encoding amino acid sequences shown in SEQ ID NOS: 2, 4 and 6, bytechniques in common use at the time of the filing date of the presentapplication, for example site-directed mutagenesis (Zoller et al.,Nucleic Acids Res. 10: 6487-6500, 1982.

Since the genes of the present invention encode enzymes which areassociated with the decomposition of DBT, they can be used todesulfurize petroleum.

(2) Vector Comprising a Gene Which Encodes a Desulfurization Enzyme

The vector of the present invention comprises the above-described first,second or third gene. Such a vector can be prepared by inserting a DNAfragment containing the first, second or third gene of the presentinvention into a known vector. The vector into which the DNA fragment isinserted is determined depending on the type of host being transformed.If Escherichia coli is used as the host, the following vector canpreferably be used. It is preferable to use vectors such as pUR, pGEX,pUC, pET, pT7, pBluescript, pKK, pBS, pBC, pCAL and the like, whichcarry lac, lacUV5, trp, tac, trc, λpL, T7, rrnB or the like as a strongpromoter.

(3) Transformant Comprising a Vector Containing Genes Which Encode aDesulfurization Enzyme

The transformant of the present invention comprises a said vector. Thecells used as a transformation host may be from a plant or animal, butmicroorganisms such as Escherichia coli are more preferable. Typicalstrains include, for example, 71/18, BB4, BHB2668, BHB2690, BL21(DE3),BNN102(C600hflA), C-1a, C600(BNN93), CES200, CES201, CJ236, CSH18, DH1,DH5, DH5 α, DP50supF, ED8654, ED8767, HB101, HMS174, JM101, JM105,JM107, JM109, JM110, K802, KK2186, LE392, LG90, M05219, MBM7014.5,MC1061, MM294, MV1184, MV1193, MZ-1, NM531, NM538, NM539, Q358, Q359,R594, RB791, RR1, SMR10, TAP90, TG1, TG2, XL1-Blue, XS101, XS127, Y1089,Y1090hsdR, YK537, and the like, which are all described in Sambrook etal, Molecular Cloning A Laboratory Manual 2nd ed.

(4). Desulfurization Enzyme

The desulfurization enzymes of the present invention includes thefollowing three proteins.

The first protein is a protein represented by an amino acid sequenceshown in SEQ ID NO: 2, or a protein comprising a deletion, substitutionor addition of one or more amino acids in the amino acid sequence shownin SEQ ID NO: 2, and having a function of converting DBTO2 into2-(2′-hydroxyphenyl) benzenesulfinic acid.

The second protein is a protein represented by an amino acid sequenceshown in SEQ ID NO: 4, or a protein comprising a deletion, substitutionor addition of one or more amino acids in the amino acid sequence shownin SEQ ID NO: 4, and having a function of converting2-(2′-hydroxyphenyl) benzenesulfinic acid into 2-HBP.

The third protein is a protein represented by an amino acid sequenceshown in SEQ ID NO: 6, or a protein comprising a deletion, substitutionor addition of one or more amino acids in the amino acid sequence shownin SEQ ID NO: 6, and having a function of converting DBT into DBTO2.

The said first, second and third proteins have a certain homology to thedesulfurization enzyme DszA, DszB or DszC derived from Rhodococcus sp.IGTS8 strain, and their function as an enzyme is also identical.However, they are apparently distinct in respect of the following.

(1) DszA, DszB and DszC cannot desulfurize benzothiophene which is adesulfurization-resistant substance, but the first, second and thirdproteins of the present invention can do so.

(2) DszA, DszB and DszC have the desulfurization activity at aroundroom-temperature region, but the first, second and third proteins haveactivity at a high-temperature region.

The desulfurization enzymes of the present invention can be prepared byusing the genes encoding the said desulfurization enzymes of the presentinvention. Further, the desulfurization enzymes represented by aminoacid sequences as shown in SEQ ID NOS: 2, 4 and 6 can also be preparedfrom the strains Paenibacillus sp. A11-1 (which was deposited with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology under accession No. FERM BP-6025 onJul. 22, 1997) or Paenibacillus sp. A11-2 (which was deposited with thesame international depositary authority under accession No. FERM BP-6026on Jul. 22, 1997) according to the conventional methods.

The characteristics of one example of the first protein of the presentinvention are as follows:

(i) Function: the first protein converts DBTO2 into 2-(2′-hydroxyphenyl)benzenesulfinic acid;

(ii) pH: as shown in FIG. 6, optimum pH: 5.5, stable pH: 5-10;

(iii) Temperature: as shown in FIG. 7, optimum temperature: 45° C.;

(iv) Molecular weight: 120,000 (as determined by gel filtration);

(v) Inhibition of activity: the first protein is inhibited by chelatingagents or SH inhibitors, but not by 2-HBP or sulfate; and

(vi) Requirement for coenzyme: NADH and FMN are required, NADPH can besubstituted for NADH, but FAD cannot be substituted for FMN.

The characteristics of one example of the second protein of the presentinvention are as follows:

(i) Function: the second protein converts 2-(2′-hydroxyphenyl)benzenesulfinic acid into 2-HBP;

(ii) pH: as shown in FIG. 8, optimum pH: 8, stable pH: 5.5-9.5;

(iii) Temperature: as shown in FIG. 9, optimum temperature: 55° C.;

(iv) Molecular weight: 31,000 (as determined by gel filtration)

(v) Inhibition of activity: the second protein is inhibited by chelatingagents or SH inhibitors, but not by 2-HBP or sulfate; and

(vi) Requirement for coenzyme: no coenzyme is required.

(5) Gene Encoding Transposase

The transposase genes of the present invention encodes any of thefollowing proteins:

(a) a protein represented by an amino acid sequence as shown in SEQ IDNO: 8,

(b) a protein represented by an amino acid sequence as shown in SEQ IDNO: 9, or

(c) a protein comprising a deletion, substitution or addition of one ormore amino acids in the amino acid sequence shown in SEQ ID NO: 8 or SEQID NO: 9, and having a transposase activity.

Among the transposase genes of the present invention, the ones encodingamino acid sequences set forth in SEQ ID NOS: 8 and 9 have beendetermined, as shown in SEQ ID NO: 7. So such genes can also be obtainedby synthesizing appropriate primers on the basis of the determinedsequence and carrying out PCR using, as a template, DNA prepared fromPaenibacillus sp. A11-1 strain (which was deposited with the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology under accession No. FERM BP-6025 on Jul. 22,1997) or A11-2 strain (which was deposited with the same internationaldepositary authority under accession No. FERM BP-6026 on Jul. 22, 1997).

The gene encoding an amino acid sequence comprising a deletion,substitution or addition of one or more amino acids in the amino acidsequence of SEQ ID NO: 8 or NO: 9 can be obtained by modifying the geneswhich encode an amino acid sequence shown in SEQ ID NO: 8 or NO: 9,according to the conventional art as of the filing date of the presentapplication, e.g. site-directed mutagenesis (Zoller et al., NucleicAcids Res. 10: 6487-6500, 1982)

Since this gene has transposase activity, it is possible to transfer anygene unit from a certain DNA molecule to a different DNA molecule byusing this gene. By the way, it has not experimentally been determinedthat the polypeptide represented by an amino acid sequence as shown inSEQ ID NO: 8 or NO: 9 has transposase activity. However, there seems tobe an extremely high possibility that each of the two polypeptide hastransposase activity for the reasons that they have a certain homologyto transposase existing in an insertion sequence IS1202, that ORFs oftwo polypeptides are in the reverse orientation to ORFs ofdesulfurization enzymes and are in a position directed to sandwich them(a structure specific for transposon), and that the direct repeatsequence (DR) and the invert repeat sequence (IR) which are specific fortransposon exist at each end of SEQ ID NOS: 8 or 9.

(6) Transposase

The transposase of the present invention is selected from the groupconsisting of:

(a) a protein represented by the amino acid sequence as shown SEQ ID NO:8,

(b) a protein represented by the amino acid sequence as shown SEQ ID NO:9, and

(c) a protein comprising a deletion, substitution or addition of one ormore amino acids in the amino acid sequence shown in SEQ ID NO: 8 or SEQID NO: 9, and having a transposase activity.

The transposase of the present invention can be prepared by using thegenes encoding the above-described transposase.

EXAMPLES

The present invention will be illustrated in more detail by the examplesdescribed below.

The experiments related to genetic engineering in the examples werecarried out mainly according to the methods described in Sambrook, J.,Fritsch, E., F. and Maniatis, T. (1989). Molecular Cloning. A laboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Example 1 Cloning of the Gene Fragment Encoding Desulfurization Enzyme

The amino acid sequences of the amino termini of both a protein havingan activity which converts DBTO2 into 2-(2′-hydroxyphenyl)benzensulfinic acid (called “protein A” hereinafter) and a proteinhaving an activity which converts 2-(2′-hydroxyphenyl) benzensulfinicacid into 2-HBP (called “protein B” hereinafter), purified fromPaenibacillus sp. A11-2 strain, were determined. The sequences are asfollows.

Protein A   NH2-MXQMXLAGFFAAGNVTXXXGA-----COOH ProteinB   NH2-TKSAIGPTRVAYSNXPVANXL-----COOH (Amino acids are expressed as aone-letter symbol. X means not yet identified.)

A homology was found between the amino acid terminal sequences of thesetwo proteins and the ones of DszA and DszB proteins encoded by dszoperon of the mesophile desulfurization bacterium, Rhodococcus sp. IGTS8strain.

Paenibacillus sp. A11-2 strain Protein A MXQMXLAGFFAAGNVTXXXGARhodococcus sp. IGTS8 strain DszA MTQQTQMHAGFFSAGNVTHAHGA Paenibacillussp. A11-2 strain Protein B TKSAIGPTRVAYSNXPVANXL Rhodococcus sp. IGTS8strain DszB GSELDSAIRDT-LTYSNCPVPNAL

Regarding Rhodococcus sp. IGTS8 strain, it is known that the 3′-terminusof the coding sequence of dszA overlaps the 5′-terminus of dszB, and dszA and dsz B are translated in different frames. Regarding the genesequence encoding the enzymes associated with the desulfurization ofDBT, it is presumed that there is some similarity between Paenibacillussp. A11-2 strain and Rhodococcus sp. IGTS8 strain. Hence, using a codingstrand of the 5′-terminal side sequence of dszA which is expected to beupstream as a sense strand and a complementary strand of the 5′-terminalside sequence of dszB which is expected to be downstream as an antisensestrand, firstly amplification of a DNA fragment containing the entiredszA was attempted.

First of all, according to the above amino acid sequences, a total offour kinds of sense primers corresponding to the amino terminalsequences of protein A and a total of four kinds of antisense primerscorresponding to the amino terminal sequences of protein B were designedand synthesized. The nucleotide sequences of all the primers are asfollows.

Sense Primers

DSZA-MIX 5′-GGN TTY TTY GCN GCN GGN AAY GTN AC-3′ THDSA-SM3 5′-TTY GCNGCN GGN AAY GT-3′ THDSA-SM4 5′-TTY TTY GCN GCN GGN AA-3′ THDSA-SM55′-GCN GGN TTY TTY GCN GC-3′

Antisense Primers

THDSB-AM2 5′-TAN GCN ACY CTN GTN GGN CCD ATN GC-3′ THDSB-AM3 5′-TAN GCNACY CTN GTN GG-3′ THDSB-AM4 5′-TCR TTN ACN GCN GTY TC-3′ THDSB-AM55′-ACY CTN GTN GGN CCD AT-3′

After combining the sense primers with the antisense primers indifferent sets, PCR was carried out, using the DNA extracted fromPaenibacillus sp. A11-2 strain as a template. The preparation of DNAfrom Paenibacillus sp. A11-2 strain was carried out as follows.Paenibacillus sp. A11-2 strain cultured in medium A containing DBT(regarding the composition, see the table set forth below) for 24 hoursat 50° C. was cultured in medium A containing fresh DBT for 24 hours at50° C. to collect the cultured cells. The obtained cells were suspendedin 1 ml of B1 buffer (50 mM EDTA, 50 mM Tris-HCl, 0.5% Triton X-100,0.2mg/ml RNaseA, pH 8.0). To this suspension, 20 μl of lysozyme solution(100 mg/ml) and 45 μl of Proteinase K solution (20 mg/ml) were added,and the suspension was reacted for 10 minutes at 37° C. After adding0.35 ml of B2 buffer (800 mM guanidine hydrochloride, 20% Tween-20, pH5.5), the reaction solution was mixed with the buffer while stirring,reacted for 30 minutes at 50° C., stirred by a mixer for 5 seconds toprepare the reaction solution of the cells. After a negativeion-exchange resin-filled QIAGEN GENOMIC-TIP20/G column (QIAGEN) wasequilibrated with 2 ml of QBT buffer (750 mM NaCl, 50 mM MOPS, 15%ethanol, 0.15% Triton X-100, pH 7.0), the reaction solution of the cellswas applied to the column. After washing the column with 3 ml of QCbuffer (1.0M NaCl, 50 mL MOPS, 15% ethanol, pH 7.0), the genomic DNA waseluted with 2 ml of QF buffer (1.25M NaCl, 50 mL Tris-HCl, 15% ethanol,pH 8.5). After 1.4 ml of isopropanol was added to the genomic DNAsolution to precipitate DNA, the obtained DNA was collected by windingaround a glass rod. The collected DNA was dissolved in 50 μl of TEbuffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to prepare a genomic DNAsolution.

TABLE 2 Composition of medium A: Glucose 5.0 g KH₂PO₄ 0.5 g K₂HPO₄ 4.0 gNH₄Cl 1.0 g MgCl₂.6H₂O 0.1 g NaCl 0.01 g CaCl₂ 0.02 g Metal solution 10ml Vitamins mix 1 ml Distilled water to 1 L pH 7.5 Metal solutionFeCl₂.4H₂O 0.5 g ZnCl₂ 0.5 g MnCl₂.4H₂O 0.5 g CuCl₂ 0.05 g Na₂MoO₄.2H₂O0.1 g Na₂WO₄.2H₂O 0.05 g Conc. HCl 10 ml Distilled water to 1 L Vitaminsmix Calcium pantothenate 400 mg Inositol 200 mg Niacin 400 mgp-aminobenzoate 200 mg pyridoxine-HCl 400 mg vitaminB₁₂ 0.5 mg Distilledwater to 1 L

The conditions of PCR wherein the prepared DNA of Paenibacillus sp.A11-2 strain was used as a template are as follows.

Compositions of the Reaction Solution

50 mM KCl 1.5 mM MgCl₂ 0.2 mM each dNTP Mixture 0.2 μM Sense primer 0.2μM Antisense primer 200 ng Template DNA 2.5 U Taq DNA polymerase

Annealing Temperature: PCR was Carried out Varying Temperatures in TwoDegrees Intervals from 44° C. to 66° C.

PCR cycle:   95° C. 1 min once   95° C. 1 min ↓ 44-66° C. 1 min repeatedfor 30 cycles   72° C. 5 min ↑   72° C. 7 min once

DNA Amplifier: Robocycler™ GRADIENT96 Temperature Cycler (STRATAGENE)

As a result of the PCR under the above conditions, it was determinedthat an amplified fragment of approximately 1.6 kb is obtained byseveral combinations of primers, when the annealing temperature is44-50° C. This 1.6 kb PCR product was cloned into Escherichia coliXL1-Blue MRF-Kan^(r) strain by using pCR-Script SK(+) vector. Bysequencing a part of the cloned DNA fragment, it was found that the 1.6kb DNA fragment contains nucleotide sequences which can encode aminoacid sequences of the amino termini of the purified protein A andprotein B. However, the sequence of the amplified DNA fragment containsa sequence which is further downstream of the nucleotide sequenceencoding amino terminus of protein B, which corresponds to thenucleotide sequence used as an antisense primer. By analyzing thedetermined nucleotide sequence, it was found that the 3′-terminal sidesequence consists of a complementary nucleotide sequence to the senseprimer corresponding to the amino terminal sequence of protein A. Thus,it was confirmed that the 1.6 kb DNA fragment was amplified as a resultof annealing the sense primer corresponding to the amino terminussequence of protein A with the nucleotide sequence downstream of thenucleotide sequence encoding the amino terminal sequence of protein B;the sense primer acted as an antisense primer.

After deducing an amino acid sequence encoded by the determined DNAsequence, this sequence was compared with each amino terminal sequenceof DszA and DszB among the proteins encoded by dsz genes cloned fromRhodococcus sp. IGTS8 strain. As a result, it was determined that thededuced sequence has a significant homology with both DszA and DszBsequences (respectively 73%, 61%). Since the homology with dsz operonDNA sequence for desulfurization genes of Rhodococcus sp. IGTS8 wasfound, we tried to further clone another DNA sequence adjacent to theDNA sequence cloned from Paenibacillus sp. A11-2 strain, using that DNAsequence as a probe.

Example 2 Preparation of the Total DNA Library

The method for preparing the total DNA is the same as the one for theDNA used as a template in PCR.

Method for Preparing the Library

The total DNA library from Paenibacillus sp. A 11-2 strain was preparedas follows. Approx. 2 μg of the total DNA sample of Paenibacillus sp.A11-2 strain was digested with 0.1 unit of Sau3AI for respectively 20,30 and 40 minutes, extracted with phenol-chloroform, and precipitatedwith ethanol to yield the digest. After centrifuging, the obtained DNAfragment was treated with 8 units of alkaline phosphatase derived fromcalf small intestine for 60 minutes at 37° C. to remove phosphoric acid.After treating with alkaline phosphatase, DNA was extracted withphenol-chloroform, and precipitated with ethanol to yield theprecipitate. Approx. 0.2 μg of the obtained DNA fragment was reactedwith approx. 2 μg of λDASHII/BamHI arm in the presence of 2 units of T4DNA ligase for 18 hours at 4° C. In vitro packaging was carried out byreacting the mixture with Gigapack II XL packaging Extract to prepare aphage library. After packaging, the titer of the phage suspension was2×10⁶ pfu.

Example 3 Screening of the Total DNA Library

A DNA probe used for the screening of phage library was prepared asfollows. As described in Example 1, there is homology between thenucleotide sequence of DNA of Paenibacillus sp. A11-2 strain, which isconsidered to encode protein A having an activity of converting DBTO2into 2-(2′-hydroxyphenyl) benzensulfinic acid and protein B having anactivity of converting 2-(2′-hydroxyphenyl) benzensulfinic acid into2-HBP, and dsz gene sequence of Rhodococcus sp. IGTS8 strain. Selecting5′ terminal side sequence of dszA of Rhodococcus sp. IGTS8 strain (from120^(th) nucleotide to 137^(th) nucleotide), whose homology isrelatively high, as a sense strand, and selecting a complementary strandto the sequence from 169^(th) nucleotide to 185^(th) nucleotide of 5′terminal of dszB coding sequence as an antisense strand, PCR primerswere prepared. By carrying out PCR with these primers and with the DNAprepared from Paenibacillus sp. A11-2 strain as a template, the sequenceof the region encoding protein A was amplified. Using the obtained PCRproduct as a template, DSZA probe labeled with dioxygenin (DIG) wasprepared by the random-prime (multi-prime) method. The preparation ofDIG-labeled probe was carried out according to the protocol ofBoehringer Mannheim. The method for preparing DIG-labeled probe is shownbelow.

1 μg (5 μl) of the obtained PCR product was denatured in boiled waterfor 10 minutes, then cooled on ice containing salt. To the obtaineddenatured DNA solution, 10 μl of hexanucleotide mixed solution (0.5MTris-HCl, 0.1M MgCl₂, 1 mM Dithioerythriol, 2 mg/ml BSA, 3.143 mg/mlRandom Primer, pH7.2), 10 μl of dNTP label mixed solution (1 mM dATP, 1mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM DIG-dUTP, pH7.5), 70 μl ofsterile distilled water and 5 μl of Klenow enzyme (10 units) were added,then reacted for 18 hours at 37° C. 5 μl of 0.5M EDTA solution was addedto the reaction mixture to stop the reaction. Then, 5 μl of 8M LiCl and275 μl of cold ethanol (−20° C.) were added, left for 30 minutes at −80°C., and centrifuged for 30 minutes at 15,000 rpm to precipitate DNA. Theprecipitated DNA was washed with cold 70% (w/v) ethanol and driedaspiration, then it was dissolved in 50 μl of TE buffer to yield a DIGlabeled probe.

The screening of protein A gene was carried out by plaque hybridizationto the plaque transferred to Hybond N+ membrane, using the DIG labeledprobe prepared by the above-described method. To detect the hybridizedclone, DIG-ELISA (Boehringer Mannheim) was used. Screening approx. 2,000phage plaques out of the genomic library by using DSZA probe, 6 positiveplaques were detected. These 6 plaques were subjected to single plaqueseparation followed by the plaque hybridization once again, whereby 4positive plaques were detected. Phage clones were prepared by using thedetected DSZA probe positive plaques, then phage DNA was extracted fromthose clones by using QIAGEN Lambda kit. The phage DNA prepared with 4positive plaques was cleaved with EcoRI, NotI, HindIII and SalI tocreate a restriction enzyme map as shown in FIG. 1. Furthermore, usingthe DSZA probe, Southern blot analysis was carried out for the DNAobtained by digesting 4 kinds of phage DNAs with EcoRI, NotI, SalI, orNotI and SalI. As a result, it was confirmed that No. 2 and No. 4 cloneswere hybridized to approx. 2 kb of NotI-SalI fragment. However,regarding No. 3 and No. 6 clones, no hybridization was observed. Basedon the results of the restriction enzyme map and Southern blot analysis,it was considered that approx. 6 kb deletion and recombination occurredin No. 3 and No. 6 phage DNAs and that dsz genes were encoded in anapprox. 8.7 kb EcoRI-HindIII fragment of No. 4 phage DNA. To examine theability to decompose DBT of Escherichia coli having each of thesubcloned DNAs, the following culture was carried out. Escherichia coliXL1-Blue having sub-cloned DNAs was cultured for a week at 37° C. in themedium prepared by adding 50 μg of yeast extract to M9 medium (Sambrooket al., Molecular cloning A Laboratory Manual 2^(nd)), followed byadding DBT, DBTO2, sodium sulfate or the like as a sulfur source. As acontrol strain, XL1 Blue strain having only vector pBluescript II KS(+)was cultured under the same conditions. Preculturing was performed in LBmedium (described in the said reference, Sambrook et al., Molecularcloning A Laboratory Manual 2^(nd)) overnight at 37° C. The cells werecollected by centrifuging the obtained preculture broth, then washedwith 66 mM of phosphate buffer, and suspended in M9 modified medium (inwhich sulfate in the M9 medium was substituted by chloride). The cellsuspension {fraction (1/100)} volume was added to an assay medium(prepared by adding DBT or DBTO2 as a sulfur source to M9 modifiedmedium), the mixture was cultured for 48 hours at 37° C. Then, thedecomposition product was extracted in accordance with standardtechniques, and gas chromatography was carried out on the product. As aresult, it was determined that regarding No. 4 clone, 2-HBP wasgenerated when the No. 4 clone was cultured in the medium containing DBTor DBO2 as sole sulfur source. However, the host XL1 Blue strain did nothave such convertion activity at all. Therefore, it was proved that thecloning DNA of No. 4 clone has a sequence which can encode the entireactivity of catalyzing the conversion reaction of DBT into 2-HBP.

Next, in order to determine the nucleotide sequence of the entire clonedDNA derived from the Paenibacillus sp. A11-2 strain, a series ofdeletion DNAs was prepared. Approx. 0.2 μg of DNA prepared from the DSZAprobe positive phage clone No. 4 was double-digested using EcoRI andHindIII, and the generated double digest was electrophoresed to purifythe approx. 8.7 kb insertion DNA fragment. After ligating this fragmentto the double digest which was obtained by treating pBluescript II KS(+) with EcoRI and HindIII and then dephosphorylated, Escherichia coliXL1 Blue strain was transformed by using the obtained hybrid DNA.Restriction enzyme analysis was carried out for the obtained subclone(p4EH), and it was determined that restriction sites KpnI and SacI didnot exist in the insertion fragment. So, to prepare a deletion plasmidused for sequencing of this insertion fragment, a combination of doubledigestions, KpnI-HindIII or SacI-EcoRI, was used, on the other hand thedeletion was carried out by actions of exonuclease III, Mung beannuclease and Klenow fragment. More specifically, the DNA fragmentobtained by cleaving subcloned DNA with SacI and EcoRI for sequencingof + strand and the DNA fragment obtained by cleaving it with KpnI andHindIII for sequencing of − strand were used, treated by exonucleaseIII, then treated by Mung Bean Nuclease and Klenow fragment of DNApolymerase I to prepare a series of deletion mutant DNAs. The sequencingreaction of the deletion mutant clone was carried out by ThermoSequenase (Amersham) and the nucleotide sequence was determined byALFred (Pharmacia). The obtained data regarding the nucleotide sequencewas analyzed by GENETYX-MAC/ATSQ v3.0 and GENETYX-MAC/ATSQ v8.0.

Subsequently, in order to determine the nucleotide sequence upstream (ordownstream of transposase) of the cloned desulfurization enzyme genesderived from Paenibacillus sp. A11-2, a series of deletion DNAs wasprepared. The digest obtained by digesting approx. 0.2 μg of DNAprepared from DSZA probe positive phage clone No. 2 with NotI and thedigest obtained by treating pBluescript II KS(+) with NotI anddephosphorylated were litigated, and then Escherichia coli JM109 strainwas transformed with the obtained hybrid DNA. After separating 20 singlecolonies, plasmid DNAs were extracted from the transformants andrestriction-analyzed by NotI treatment to obtain subclones pBS2N2 andpBS2N3 into which an approx. 3 kb of NotI fragment was inserted. ThepBS2N2 and pBS2N3 are subclones wherein the 3 kb NotI fragment wasinserted in the reverse direction to each other. Regarding pBS2N2 andpBS2N3, a series of deletion DNAs was prepared by using KpnI, HpaI,NruI, PstI and XhoI. The sequencing reaction of deletion clone wascarried out by Thermo Sequenase (Amersham) and the nucleotide sequencewas determined by ALFred (Pharmacia). The obtained data regarding thenucleotide sequence was analysized by GENETYX-MAC/ATSQ v3.0 andGENETYX-MAC/ATSQ v8.0.

Analyzing ORF in the determined sequence indicated existence of threeORFs whose length was more than 1 kb in the center of 8.7 kb of theinserted DNA. These ORFs were named ORF1, ORF2 and ORF3 from 5′ side. Inaddition to them, there existed one homologous ORF in the vicinity ofeach end of the inserted DNA. ORF1, ORF2 and ORF3 respectively encode454, 353 and 414 amino acids. It was determined that the terminationcodon TGA of ORF1 and the initiation codon ATG of ORF2 are partiallyoverlapped, and the overlapped sequence is 5′-ATGA-3′ which has the samestructure as the nucleotide sequence in the dsz operon of IGTS8. Whenanalyzing the nucleotide sequence homology between these ORFs and dszgenes of IGTS8 strain, ORFs 1, 2 and 3 respectively showed approx. 64%,54% and 48% of homology with dsz A, B and C of IGTS8 strain. Inaddition, when deducing the amino acid sequences of the proteins encodedby the nucleotide sequence of Paenibacillus sp. A11-2, the polypeptidesencoded by ORFs 1, 2 and 3 respectively showed 65%, 54% and 52% ofhomology with DszA, DszB and DszC of IGTS8 strain.

Comparing the amino acid sequence of the protein encoded by ORF ofPaenibacillus sp. A11-2 strain with that encoded by the dsz sequence ofRhodococcus sp. IGTS8, characteristic differences were found in severalpoints. First, regarding protein A encoded by ORF1 and DszA, theirsequences at the amino terminus and the carboxyl terminus are completelydifferent, standing in sharp contrast to the internal amino acidsequences whose homology is relatively high. Second, protein A haslonger amino and carboxyl termini. On the other hand, the amino acidsequences of protein B encoded by ORF2 and DszB are completely differentfrom the relationship between protein A and DszA; the amino and carboxyltermini of DszB extend longer than both termini of protein B, and aboveall, homology is not found in the amino terminal sequence. Comparing theamino acid sequences of protein C encoded by

ORF3 and DszC, then full lengths are almost the same, but the sequencesof the amino terminal sides are completely different.

In approx. 8 kb DNA whose nucleotide sequence was determined, one ORFwas found upstream of a series of sequences of ORF1, ORF2 and ORF3, andtwo ORFs were found downstream. The lengths of the upstream ORF and themost downstream ORF are both approx. 1 kb, they show a perfect homology,and the polypeptides encoded by the ORFs was determined to have approx.30% homology at the amino acid level to the transposase in the insertionsequence IS1202. The ORF encoding this transposase was oriented in thereverse direction to the ORF for desulfurization gene. The fact that aseries of ORFs encoding desulfurization activity was sandwiched by theinsertion sequence-like sequences suggested the possibility that theseDNA sequences form a sort of transposon. Moreover, it was also detectedthat approx. 0.6 kb ORF, which was found between the insertionsequence-like sequence positioned at the most downstream and a series ofORFs encoding desulfurization activity, encoded the amino acid sequencewhich showed approx. 40% homology with carbonic anhydrase.

Example 4 Separation of Desulfurization-ability Deficient StrainPaenibacillus sp. M18 and Analysis of its Properties

Paenibacillus sp. A 11-2 strain was treated with acridine orange so thatthe mutant strain M18 which lost the ability to decompose DBT wasseparated. First, A11-2 strain was cultured in 2×YT medium overnight at50° C., and 0.1 ml of the obtained overnight-cultured broth wastransferred into 5 ml of 2×YT medium containing 30 μg/ml of acridineorange, then it was cultured overnight at 50° C. The cells werecollected by centrifugation and washed once with medium A. The washedcells were suspended in 0.1 ml of medium A, then transferred into 2 mlof 2×XY medium and cultured for four hours at 50° C. The cultured brothwas applied to a 2×YT agar medium and cultured overnight at 50° C. Thegenerated colony was transferred into medium A whose sulfur source wasonly DBT, its ability to utilize DBT was detected and finally adesulfurization deficient strain (M18 strain) which cannot utilize DBTwas obtained. The fact that the mutant strain M18 lost the activity ofdecomposing DBT was confirmed by culturing the said strain in a mediumcontaining DBT and various methyl DBT derivatives and analyzing itsgrowth. After collecting cells from M18 strain and its parent strainwhich were cultured in AYD medium overnight, those cells were washedwith AY medium two times, then were suspended in AY medium. 5 ml of AYmedium was contained in a screw capped test tube, on which 1 ml ofn-tetradecane containing 50 ppm in sulfur concentrations of each organicsulfur compound was layered, then 100 μl of the cell suspension preparedby the above-stated method was added, and it was cultured for a day at50° C. After the culture, 100 μl of 6N hydrochloric acid was added, wasstirred, and was extracted with 1 ml of ethyl acetate. Finally gaschromatography and gas chromatography/mass spectrometry were carried outto the obtained ethyl acetate-n-tetradecane layer. As a result, it wasdetermined that, for any of the detected organic sulfur compounds, M18strain cannot use them as only sulfur sources and does not show afeature of decomposing them. In the case of a room-temperaturedesulfurizing strain Rhodococcus sp. IGTS8, DBT is decomposed over apath such as DBT→DBTO→DBTO→2-(2′-hydroxyphenyl) benzenesulfinicacid→2-HBP+sulfite (Oldfield, C., Pogrebinsky, O., Simmonds, J., Olson,E. S. and Kulpa, C. F. Microbiology, 143:2961-2973, 1997). It is knownthat 2-(2′-hydroxyphenyl) benzenesulfinic acid provides DBT sultine whenit forms a ring (Olson, E. S., Stanley, D. C. and Gallagher, J. R.Energy & Fuels 7:159-164, 1993). Further, it has been reported that,because of the enzyme activity of DszA, Rhodococcus sp. IGTS8 strain, inassociation with reductase, converts DBT sultone into 2-HBP and sulfite(Oldfield, C., Pogrebinsky, O., Simmonds, J., Olson, E. S. and Kulpa, C.F. Microbiology, 143:2961-2973, 1997). Using a medium containing theintermediate metabolite of this pathway as the only sulfur source, theavailability and bioconversion of the sulfur source by M18 strain werestudied. The result is that the strain could not use any of DBTO, DBTO2,DBT sultine and DBT sultone as the sulfur source, and conversionactivity was not detected either. Taking this result into account, it isconsidered that M18 strain has lost a whole series of enzyme activityinvolved in the decomposition reaction pathway wherein DBT is decomposedinto 2-HBT.

Example 5 Proof of the Desulfurization Activity of the Protein Encodedby ORF in Recombinant DNA

In order to determine that a cloned DNA is the genetic entity whichexpresses desulfurization activity, that is, the activity of decomposingDBT, a recombinant plasmids were prepared such that a sequencecontaining, a DNA fragment with all or part of ORF1, 2 and 3 waspositioned downstream of Ptac, a strong promoter acting in Escherichiacoli, and then Escherichia coli, JM109 strain was transformed with eachof the obtained recombinant plasmids. The detailed method for preparingvarious recombinant plasmids is described below. First, 8.7 kbEcoRI-HindIII fragment derived from Paenibacillus sp. A11-2 strain DNAwas cloned into phagemid vector pBluescript II KS(+) to obtain arecombinant DNA p4EH which was then double-digested with ClaI and SmaIthereby obtaining a ClaI-HindIII fragment. Similarly, pBluescript IIKS(+) was cut with ClaI and HindIII to recover a larger fragment. Thislarger fragment was subsequently ligated to the obtained ClaI-HindIIIfragment to prepare a recombinant DNA pB14. Second, pB14 wasdouble-digested with XbaI and KpnI, and a DNA fragment containing theentire DNA derived from the cloned Paenibacillus sp. A11-2 strain wascollected and ligated to the larger fragment which was obtained bydouble-digesting pHSG298 plasmid with XbaI and KpnI, thereby to preparerecombinant DNA pSKR6. This pSKR6 was double-digested with EcoRI andHindIII, and was inserted into EcoRI-HindIII site of expression vectorpKK223-3 to prepare expression plasmid pSKR7. Escherichia coli JM109strain was transformed with this pSKR7 to obtain transformant strain#121 (pSKR7). In this strain, there are approx. 50 bp between ATGsequence which seemingly corresponds to the initiation codon of ORF1which is presumed to correspond to dszA on the most 5′ side of dszoperon of IGTS8 strain and Shine-Dalgarno (SD) sequence disposeddownstream of the expression promoter Ptac on pKK223-3. Experiments onthe expression of genes from various Escherichia coli and foreign geneshave indicated that the distance between the SD sequence and the ATGinitiation codon has a very large influence over the translationefficiency of the gene (e.g. Horwich, A, Koop, A. H. and Eckhart, W.Mol. Cell. Biol. 2:88-92, 1982; Gheysen, D., Iserentant, D., Derom, C.and Fiers, W. Gene 17:55-63, 1982). So, in order to shorten the distancebetween the SD sequence and the ATG initiation codon, plasmid pSKR7 wascleaved at ClaI site immediately followed by ORF of dszA (5′-ATCGAT-3′;G being on the 3′ side forms the sequence of the ATG initiation codon)and at EcoRI site, the generated cohesive terminus was treated withT4DNA polymerase to be blunt-ended, and a ring-closure was done again byligation. By carrying out this treatment, the distance between the SDsequence and the ATG initiation codon was shortened to 11 bp. Now,Escherichia coli JM109 was transformed with this recombinant plasmid,and the obtained transformant strain was named #361 strain.

6 ml of LB-Amp-DBT medium (containing 10 g of Bacto polypeptone, 5 g ofBacto yeast extract, 10 g of NaCl, 50 mg of Ampicillin, 100 mg of DBT in1 L) was contained in each of screw capped test tubes whose diameter is18 mm, 1 % of #361 strain suspension cultured overnight on the samemedium was inoculated, then it was cultured at 37° C. Every two hoursafter the beginning of the culture, two test tubes were taken out, andthe entire cultured broth of each test tube was extracted with 1.2 ml ofethyl acetate and was analyzed and quantified by gas chromatography.Also the turbidity of the cultured broth was measured byspectrophotometer every two hours after the beginning of the culture.Consequently, it was confirmed that DBT was decreasing while culturedfor 4-8 hours and that 2-HBP being the metabolite of DBT was generatedin the medium. FIG. 3 shows the decrease of DBT and the formation of DBTmetabolite in this medium, wherein each numerical value represents theaverage analytical value obtained from the two test tubes. Since DBTremarkably decreased for 4-6 hours after the beginning of the culture,we intended to analyze the activity of the cell free extraction systemusing the cells cultured for 6 and 8 hours.

The preparation of cell free extracts was carried out as follows. To 100ml of LB medium (LB-Amp medium) containing 50 mg/ml of Ampicillin, 1 mlof overnight-cultured broth of #361 strain prepared from the same brothwas inoculated, and then it was cultured for 6 or 8 hours at 37° C.After collecting and washing the cultured cells, they were suspended inTH buffer (50 mM Tris-HCl, 1 mM PMSF, 10% glycerol, pH7.0) so that OD₆₆₀becomes 25. The cell suspension was treated by an ultraoscillator for 10minutes two times, and the obtained cell suspension was centrifuged at11,000 rpm for 60 minutes to prepare cell free extracts. The reaction ofthe cell free extracts system was carried out as follows. To 0.7 ml ofthe prepared cell free extracts, 0.3 ml of cell free extracts preparedfrom the mutant strain M18 of Paenibacillus sp. A11-2 which does nothave desulfurization activity in the same manner as stated above, 3mM ofNADH, 10 μM of FMN and approx. 50 ppm of DBT were added, then thereaction was carried out by rotary-shaking for four hours at 37° C. or50° C. The obtained reaction mixture was extracted in accordance withstandard techniques and DBT and DBT metabolite were analyzed by gaschromatography. In addition, using a portion of the cell suspensionprepared so that OD₆₆₀ was adjusted to 25, a resting cell reaction wasalso carried out. Regarding the resting cell reaction, approx. 50 ppm asthe final concentration of DBT was added to 1 ml of the cell suspensionfollowed by the reaction for five hours at 37° C. The obtained reactionmixture was analyzed by gas chromatography in accordance with standardtechniques.

FIG. 4 shows the result of the reactions carried out at 37° C. and 50°C. adopting DBT as a substrate using the cell free extracts obtainedfrom the cells of #361 strain cultured for 6 and 8 hours. Regarding thecells cultured for 8 hours, the activity of decomposing DBT in a restingcell reaction system which was examined concurrently is also disclosed.As shown in FIG. 4, it was observed that in the reactions at 37° C. ofboth the cell free extracts system and the resting cell system, thereaction of generating 2-HBP using DBT as a substrate progressed, and itwas determined that both of them have desulfurization activity. Inaddition, regarding the cell free extracts system, the formation of2-HBP from DBT at 50° C., that is to say, desulfurization activity wasalso clearly confined. From this result, it was proved that the DNAfragment derived from the cloned Paenibacillus sp. A11-2 strain DNAactually carried on the activity of decomposing DBT at high temperature.On the other hand, when the cell free extracts prepared by the samemethod as for #361 strain was used, applying the parent strain JM109 andthe JM109 containing only vector pBluescript II KS(+), no 2-HBP wasgenerated at all. Moreover, with this cell free extracts of #361 strain,even at 50° C., the conversion of benzothiophene into the desulfurizedproduct o-hydroxystyrene was observed. This shows that the activity ofdecomposing benzothiophene at high temperature is also carried by theDNA of A11-2 strain introduced into Escherichia coli.

It was presumed that the DNA fragment carrying desulfurization activityderived from Paenibacillus sp. A11-2 strain contains 3 ORFs and that,considering its nucleotide sequence, it has the same gene structure asdesulfurization genes cloned from Rhodococcus sp. IGTS8 strain andRhodococcus erythropolis KA2-5-1 strain. Hence, various deletion DNAfragments were prepared using recombinant plasmids of #361 strain, andthe relation between the deletion DNA fragments and the activity of DBTdecomposition system of each ORF was analyzed. The linear DNA obtainedby cleaving #121 plasmid at BsrGI site situated 12 bp upstream of ATGinitiation codon of ORF2 and at EcoRI site downstream of SD sequence wastreated with T4DNA polymerase then T4DNA ligase to prepare a recyclizedrecombinant plasmid. After transforming Escherichia coli JM109 with thisplasmid, the obtained transformant strain containing ORF2 and ORF3 onthe cloned DNA from Paenibacillus sp. A11-2 strain was named #233.Following the same method, the transformant strain #234 containing onlyORF3 was prepared by using SacI site immediately followed by ORF3 andEcoRI site situated downstream of the SD sequence, and the transformantstrain #391 containing only ORF2 was prepared by using BsrGI site andPstI site. Furthermore, the transformant strain #401 containing ORF1 andORF2 was prepared by using PstI site situated inside of ORF3 of thetransformant strain #361 and PstI site derived from a vector. Each ofthese transformant strains having deletion DNAs was cultured in LB-Ampmedium overnight, and 50 μl of the cultured broth was inoculated upon 5ml of LB-Amp medium, into which DBT, DBTO2 or DBT-sultine were added, toobtain 50 mg/l as the final concentration, then it was culturedovernight at 37° C. The obtained overnight-cultured broth was extractedwith 1 ml of ethyl acetate, and the extract was analyzed/quantified bygas chromatography. The results are shown in Table 3.

TABLE 3 Yield (μM) Sample Contained ORF substrate DBT DBTO DBTO2 Sultine2-HBP Total Blank DBT 136 0 0 0 0 136 DBTO2 0 0 117 0 0 117 Sultine 0 00 54 9 63 vector DBT 130 0 0 0 0 130 DBTO2 0 0 117 0 0 117 Sultine 0 0 061 7 69 #361 ORF1 DBT 72 0 0 0 48 119 ORF2 DBTO2 0 0 78 0 34 112 ORF3Sultine 0 0 0 51 27 78 #233 ORF2 DBT 101 0 24 0 0 125 ORF3 DBTO2 0 0 1140 0 114 Sultine 0 0 0 55 18 73 #234 ORF3 DBT 104 0 21 0 0 125 DBTO2 0 0116 0 0 116 Sultine 0 0 0 60 9 69 #391 ORF2 DBT 126 0 0 0 0 126 DBTO2 00 117 0 0 117 Sultine 0 0 0 52 20 72 #401 ORF1 DBT 127 0 0 0 0 127 ORF2DBTO2 0 0 2 0 99 101 Sultine 0 0 0 35 44 79 #421 ORF1 DBT 126 0 0 0 0128 DBTO2 0 0 0 58 7 65 Sultine 0 0 0 56 7 63 The amount of the addedsubstrate; DBT: 136 μM, DBTO: 125 μM, DBTO2: 118 μM, Sultine: 107 μM

From the data regarding the formation of DBT metabolite by eachtransformant strain shown in the table, it is known that 3 ORFs in theDNA cloned from Paenibacillus sp A11-2 strain were associated with DBTdecomposition. First, due to the fact that DBTO02 was generated from DBTin #361, #233 and #234 but it was not so in #391, #401 and #421, it isclear that ORF3 encodes oxygenase having an activity of generating DBTO2from DBT. Second, due to the fact that DBT-sultine was generated fromDBTO2 in #361, #401 and #421, but was not so in #233, #234 and #391, itis known that ORF1 encodes oxygenase having an activity of generatingDBT-sultine from DBTO2. It was observed that a small amount of 2-HBP wasgenerated from DBT-sultine even in the control test wherein only LB-Ampmedium without cells but containing DBT-sultine as the only sulfursource was shaken in the same conditions as in the recombinant clones.The present inventors have carried out various control tests andconfirmed that this is a spontaneous reaction occurred without enzymesor cells. Consequently, it is necessary to adjust the above result bysubtracting the amount of 2-HBP more or less equal to that observed in“Blank” from each of the amounts determined using each transformantstrain. As a result of such an adjustment, 2-HBP was generated fromDBT-sultine in #361, #233, #391 and #401, but it was not so in #234 and#421. For this reason, it is known that ORF2 encodes desulfinase havingan activity of generating 2-HBP from DBT-sultine.

Example 6 Culture of Paenibacillus sp. A11-2 Strain

A medium (150 ml) having the same composition as medium A used inExample 1 was contained in a 500 ml-capacity of sealed screw cappedconical flask with a baffle, 50 mg/l of DBT and cultured broth of A11-2strain were added thereto, and it was rotary-shaken at 120 rpm at 50° C.After culturing it overnight, the cultured broth was centrifuged at5,000 rpm for 10 minutes at 4° C. to collect cells.

Example 7

(1) Purification of Protein A

The cells from Example 6 (wet weight 30 g) were suspended in buffer A(20 mM Tris-HCl, pH7.5, 10% glycerol, 1 mM dithiothreitol, 1 mMphenylmethanesulfonylfluoride) and were sonicated by an ultraoscillator(Branson, model 450) for 15 minutes at 4° C. three times. Aftercentrifugation at 5,000×g for 10 minutes to remove intact cells, thesupernatant was centrifuged at 100,000×g for 60 minutes. The obtainedsupernatant was passed through a filter whose pore size is 0.22 μm andwas applied to an anion exchange column (Pharmacia, HiLoad Q 26/10)equilibrated with buffer B (20 mM Tris-HCl, pH7.5, 10% glycerol, 1 mMdithiothreitol). After washing with buffer B, elution was carried outwith linear gradient from buffer B to buffer B containing 0.5M sodiumchloride. Active fractions (0.35-0.4M sodium chloride) were collectedand concentrated by ultrafiltration. After diluting with buffer A,ammonium sulfate was added to prepare 30% saturated solution. Thissolution was applied to a hydrophobic chromatography column (Pharmacia,HiLoad Phenyl Sepharose HP) which was equilibrated with 30% saturatedbuffer containing ammonium sulfate. Active fractions were collected,concentrated by ultrafiltration (Millipore, Ultrafree15, molecularweight 10,000 cut-off), desalinated by a desalting column (Pharmacia,PD-10), and then were applied to an anion exchange column (Bio/Rad,Proteinpack DEAE) equilibrated with buffer B. Active fractions werecollected, concentrated by ultrafiltration, desalinated by a desaltingcolumn, and then were applied to a hydroxyapatite column (Bio/Rad,BioGel HPHT) equilibrated with buffer C (10 mM potassium phosphate,pH7.1, 10% glycerol, 1 mM dithiothreitol). After washing with buffer C,elution was carried out with linear gradient from buffer C to buffer Ccontain 0.2M potassium phosphate. As a result, it was confirmed that theactive fractions were electrophoretically uniform.

(2) Measurement of Enzyme Activity

To the buffer containing 3mM of NADH and 10 μm of FMN (50 mM Tris-HCl,pH7.0) the enzyme solution was added, and further 0.4 ml of cell freeextracts of M18 strain, which does not have an ability to utilize DBT,obtained by curing treatment for A11-2 was also added. After apreincubation for two minutes at 50° C., DBTO2 solution(dimethylformamide solution)was added to obtain 50 mg/l as the finalconcentration (the total amount of solution is 1 ml). At the end of thereaction, 10 μl of 6N hydrochloric acid and 0.4 ml of ethyl acetate wereadded, fully mixed, then centrifuged at 12,000 rpm for 3 minutes. Then,analysis by gas chromatography was carried out to the obtained upperlayer (ethyl acetate layer). The specific activity is represented suchthat 1 denotes activity decomposing 1 nmol of DBT-sulfone per 1 mg ofprotein per a minute.

Enzyme activities in each step of purification are shown in Table 4 andthe activities with various pHs and temperatures are shown in FIGS. 6and 7.

TABLE 4 Specific Total Protein acitivity Activity (mg) (U/mg) (U) Crudeextract 1488  2.1 3125 HiLoad Q 26/10  144 13.3 1915 HiLoad PhenylSepharase HP  40 31.3 1252 Protein Pack DEAE   5 68.3  342 BioGel HPHT  1 100    100

Example 8

(1) Purification of Protein B

The cells from Example 6 (wet weight 13 g) were suspended in buffer A(20 mM Tris-HCl, pH7.5, 10% glycerol, 1 mM dithiothreitol, 1 mMphenylmethanesulfonylfluoride) and were sonicated by an ultraoscillator(Branson, model 450) for 15 minutes at 4° C., three times. Aftercentrifugation at 5,000×g for 10 minutes to remove intact cells, thesupernatant was centrifuged at 100,000×g for 60 minutes. The obtainedsupernatant was passed through a filter (Millipore Millex GV, pore size0.22 μm) and was applied to an anion exchange column (Pharmacia, HiLoadQ 26/10) equilibrated with buffer B (20 mM Tris-HCl, pH7.5, 10%glycerol, 1 mM dithiothreitol). After washing with buffer B, elution wascarried out with linear gradient from buffer B to buffer B containing0.5M sodium chloride. Active fractions (0.15-0.2M sodium chloride) werecollected and concentrated by ultrafiltration (Millipore, Ultrafree 15,molecular weight 5,000 cut-off). After diluting with buffer A, ammoniumsulfate was added to prepare 30% saturation. This solution was appliedto a hydrophobic chromatography column (Pharmacia, HiLoad PhenylSepharose HP) which was equilibrated with 30% saturated buffercontaining ammonium sulfate. Active fractions were collected,concentrated by ultrafiltration, desalted by a desalting column(Pharmacia, PD-10), and then were applied to an anion exchange column(Bio/Rad, Bioscale DEAE) equilibrated with buffer B. Active fractionswere collected, concentrated, desalted, and then were applied to ahydroxyapatite column (Bio/Rad, BioGel HPHT) equilibrated with buffer C(10 mM potassium phosphate, pH7.1, 10% glycerol, 1 mM dithiothreitol).After washing with buffer C, elution was carried out with lineargradient from buffer C to buffer C containing 0.2M potassium phosphateand then it was applied to an anion exchange column (Pharmacia, Mono QHR5/5) equilibrated with buffer B. After washing with buffer B, elutionwas carried out with linear gradient from buffer B to buffer Bcontaining 0.5M sodium chloride. As a result, it was confirmed that theactive fractions were electrophoretically uniform.

(2) Measurement of Enzyme Activity

Enzyme solution was added to buffer D (50 mM Tris-HCl, pH7.0), and afterpreincubation for two minutes at 50° C., sultine (in N,N-dimethylformamide) was added to obtain 50 mg/l as the finalconcentration (total volume 1 ml). At the end of the reaction, 10 μl of6N hydrochloric acid and 0.4 ml of ethyl acetate were added, fullymixed, then analysis by gas chromatography was carried out to theobtained upper layer (ethyl acetate layer). The measurement of activitywas carried out by quantifying 2-HBP produced. The specific activity isrepresented such that 1 unit denotes activity producing 1 nmol of 2-HBPper 1 mg of protein per minute. To prevent the influence of 2-HBPinhibiting the activity, sodium 2-phenylbenzensulfinate was used as asubstrate, and the activity was measured by quantifying the generatedbiphenyl.

Enzyme activities in each step of purification are shown in Table 5 andthe activities at various pHs and temperatures are shown in FIGS. 8 and9.

TABLE 5 Specific Total Protein acitivity Activity (mg) (U/mg) (U) Crudeextract 504 2.2 1109 HiLoad Q 26/10 120 10 1200 HiLoad Phenyl SepharaseHP 18 31  558 Protein Pack DEAE 7 16  112 BioGel HPHT 1 85  85 Mono Q0.2 139  28

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

Advantage of the Invention

The present invention provides novel genes and enzymes associated withdesulfurization. By using these genes and enzymes, sulfur existing infossil fuel can be easily removed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a restriction map of insert DNA in DSZ probe positiveclone.

FIG. 2 shows a construction process of expression plasmid pSKR7.

FIG. 3 shows a result of DBT decomposition by #361 strain.

FIG. 4 shows a result of DBT decomposition reaction with cell freeextracts from #361 strain.

FIG. 5 shows a structure of deletion-expression plasmid.

FIG. 6 shows a relation between temperature and the enzyme activity ofprotein A.

FIG. 7 shows a relation between temperature and the enzyme activity ofprotein A.

FIG. 8 shows a relation between pH and the enzyme activity of protein B.

FIG. 9 shows a relation between temperature and the enzyme activity ofprotein B.

21 1 9775 DNA Paenibacillus sp. CDS (3031)...(4410) 1 gcggccgcgtcatcttgccg ccgctcgatg cggtttatcc gatcaatgca aaggacgcaa 60 ttcctccttcgcattcctgc ggggtcgaac cgtatcagcc gcaacggatg atttccaatg 120 aaatggccgcgatgctgatt tcgaccgtcg tgaatgagct gttttcgtcg aacgccattc 180 tcgtccattatgtcaatttt aatgcaaaga ccgggaactg caggccggtt tatgcagaag 240 atgtggccggcgccaataac gattccgctt cggtagcagc tgcgccgtat gaccaggaag 300 ctgactccggactgcaatca agcgagagtg gccaactcca acatgatccg gacaatgctg 360 tatccccgtctacaaaagag gaggacgctg aaatcctttc tgccgaggag cttcctgcgg 420 aacaggggggcgccgaggta gaggtcccgg aaagtggagt ggccggcgtt cgggagaatg 480 gtatcagggtaattcgcatc gaaccacttg acgagaaaca cgagaagacg caacacggat 540 acggggtacctgtgctttat catctggaag acgggtccac gctccgtaag ttaattacgg 600 ggactcgactgagggacgct aaagcccgtg ttgaaaggct cagtcgcgat cctggcgacc 660 ggtggattgaacgcaccgaa aacggactcg tgattgaaaa atcgtcgatc ggtcttgtcg 720 ggtaaggaaaattgggggcg tattttatgc ccctttttct ttttttataa gggtggaaat 780 atcgcgcaagttaaggggga gcttgagcaa atgaaggtgg ataccgcaaa aattttcaag 840 aagtttaagaaggtcattga tacccgcgac atcaatcaca tggacaagca gctttacaat 900 tatttgcatcttcatgcagg cttcatcgcg cattatgaca tctatggctt caaagagaca 960 tattccgataaagggtttct tgatttcatt gagcattttg agcagtgcta ttatttgtgc 1020 tacggtgaatacggagagtt taaccgcgaa ctgaaggaat atgtgctgca acatgcggag 1080 cagatccgcgctgaatttgc ttataaggcg cagcaacatg aattgaaact gctccagaag 1140 ctggcggcaaagcacggcaa aatcatttcc gacgttgcga tgaaccaaga tcaagacatg 1200 acggctgctgtggtaccgat gtcgcttgcc gcgaacgggc aattggaatt tgcgctgtga 1260 taaatgggaagggtggagca ttccactctt cctatttatc ttttcaaatt tcggcagcat 1320 accacaattttagagttttg gttggacaat ggctgggtaa tatgtcaagc gtctgtgaaa 1380 atgtcaggttaactgttcta tgaaaatgtc agggatgata gttgattaaa cagccgccgt 1440 cctcttgcagactagccgga tgctgtgcta cgctgtaact gcttgctgga gaatggtttt 1500 ctccagggatggtttgcagc gggcttgcgg ggggacgcag gcgccgcttc ttttttggcc 1560 gttgttggcgccggggtctg tgtggcctgt gtctccacac aaggccaggc ccgcccttga 1620 tcccacagccacacttgtcc atccatgccg acacgcactt cgacgacgct cttcgcttcc 1680 cagcgcggaacaccggggac gggctttggc atgtagcatt tccctttcca gaagaacgtc 1740 tgcccgccgctgatgcgccg gtattcccga cgcgtgaaga tatgctccaa aggcgtttcg 1800 ggcagcggccggtaggccgg ttcagcttct tgcggcgcga cggcaaactg acgattgtgc 1860 ttggcgataagttccggtaa cacgcgattg gcttcctcca tcgtgcacac gttgcgcagc 1920 ctaagttcgatcaccaggcg atcctgaaag gtttgccaga gccgttcgat ccgtcctttg 1980 gcttggggtgacagcgcctc gatatgggta atgcccagat cggcgagggc ctgtccgaag 2040 gtggaaagcgacggcggctc accggccaat tcctgctcga gggttggctt gcccttgggc 2100 gggtgaaaaatggagtgttg gtcgctgtag agcgcaagcg gtacgccttt gcgcctaagt 2160 ccctcgatcatgacggtcac gtagccctcc agtgtttcgg tcgggcggaa ggtggccgcg 2220 accacttccccggtggcgtc atcgatgatg ccgtgcaggg tgagcatggg accgcgatcc 2280 tccagccaggcatagggaga agcatcgatc tgccacagca tgcccgcctg aggtttgcgg 2340 ggccggggtcggtgagcctt cggacgacgg cgcagccgcg cgggacgcaa cccgccttcc 2400 agcagaatgcggcggaccga agagacgctt aaatggatgt tttcgtgttc ggccaacagc 2460 tcggcaaagtgggtggcatt gcttccgaag tagcgctcct gatacaggag cataacgcgt 2520 tgtttgagcgaatcggtcaa ggtgtgagcc ggcttacggc cccgattccc atgtgcgatc 2580 gcttgtgcacctccgtgacg atatttggcc ttgagccgat acgcttgacg gacactgatg 2640 cccaggttgcgtgcaacatc ctgttccgtg agatggccgt cgatccattt ttcaatgacc 2700 ataacgcgtttcagttcgtt ctttgtcaag gtgatctgct ccttgctcat actgacattt 2760 tctcggatcagttacaccct gacaatatca cagaacaaca acatgagtga ttgcgacggg 2820 ttgacaaaatgaatcctgaa cggtatactc cgattcataa atactaatca atttaatcgg 2880 gtttacctcggctgactgga ccaccagagg ccctctgact ttgcggtaat tttgccggaa 2940 agcggggggctttttctttt gcagaggagg gccgaaaaac agttttctgc tcctggatga 3000 ccattgaagaacattcacgc aggaacatac atg gga ggt gtt caa tcg atg cgt 3054 Met Gly GlyVal Gln Ser Met Arg 1 5 caa atg cat ctt gcc ggt ttt ttt gca gcg ggt aatgtg acc cat cac 3102 Gln Met His Leu Ala Gly Phe Phe Ala Ala Gly Asn ValThr His His 10 15 20 cac ggg gca tgg cgt cac ccg aaa act gat aat ggt tttttg tct att 3150 His Gly Ala Trp Arg His Pro Lys Thr Asp Asn Gly Phe LeuSer Ile 25 30 35 40 tct tgg tat caa cac atc gcc cgt aca ctc gag cgc ggccgc ttt gac 3198 Ser Trp Tyr Gln His Ile Ala Arg Thr Leu Glu Arg Gly ArgPhe Asp 45 50 55 ctg ctc ttt ctg cct gac ggt ttg gct att tgg gat agc tacgga aac 3246 Leu Leu Phe Leu Pro Asp Gly Leu Ala Ile Trp Asp Ser Tyr GlyAsn 60 65 70 aat ctt gat gct gga ttg aga ttt gga ggc caa gga gcc gct tttctg 3294 Asn Leu Asp Ala Gly Leu Arg Phe Gly Gly Gln Gly Ala Ala Phe Leu75 80 85 gat ccc gtc ccc gtg ctc gcc acc atg gct gcg gcc acg gag aga ctg3342 Asp Pro Val Pro Val Leu Ala Thr Met Ala Ala Ala Thr Glu Arg Leu 9095 100 ggc ctg ggg gcc acg att tcg aca acc tac tat cct cct tac cat gtg3390 Gly Leu Gly Ala Thr Ile Ser Thr Thr Tyr Tyr Pro Pro Tyr His Val 105110 115 120 gca aga gtg ttt gct acg ctg gat cac tta aca aaa gga agg gcagcc 3438 Ala Arg Val Phe Ala Thr Leu Asp His Leu Thr Lys Gly Arg Ala Ala125 130 135 tgg aat gtc gtg acc tca ctc aac aac gcc gag gcc agg aac tttggg 3486 Trp Asn Val Val Thr Ser Leu Asn Asn Ala Glu Ala Arg Asn Phe Gly140 145 150 tat gag gaa cac ctg gat cac gat agt cgg tac gac cgt gcc gatgag 3534 Tyr Glu Glu His Leu Asp His Asp Ser Arg Tyr Asp Arg Ala Asp Glu155 160 165 ttt ctt gag att aca gat aaa ttg tgg agg agt tgg gat cag gatgca 3582 Phe Leu Glu Ile Thr Asp Lys Leu Trp Arg Ser Trp Asp Gln Asp Ala170 175 180 ttg ctc ctc gac aaa aaa cag ggt ctt ttt gct gat ccc aga aaggtc 3630 Leu Leu Leu Asp Lys Lys Gln Gly Leu Phe Ala Asp Pro Arg Lys Val185 190 195 200 cac tat att gat cac tcc gga acc tgg ttc tcc gtc cgg ggcccg tta 3678 His Tyr Ile Asp His Ser Gly Thr Trp Phe Ser Val Arg Gly ProLeu 205 210 215 caa gtc ccg cgg tcg cca cag ggt cgt cct gtc atc att caggcg gga 3726 Gln Val Pro Arg Ser Pro Gln Gly Arg Pro Val Ile Ile Gln AlaGly 220 225 230 tcc tcc gcc cgt gga aag aca ttt gct gct cgg tgg gca gaagcc gtt 3774 Ser Ser Ala Arg Gly Lys Thr Phe Ala Ala Arg Trp Ala Glu AlaVal 235 240 245 ttc acc att gcg ccg aac cga gtc gcg atg cgg gcg ttt tacgaa gac 3822 Phe Thr Ile Ala Pro Asn Arg Val Ala Met Arg Ala Phe Tyr GluAsp 250 255 260 ttg aaa aaa cag gta atc gcc gca gga cgc cgt ccc gag aattgc aaa 3870 Leu Lys Lys Gln Val Ile Ala Ala Gly Arg Arg Pro Glu Asn CysLys 265 270 275 280 ata ctc cct gcc gtc att ccg att ctt ggc gat acg gagaag gaa gcg 3918 Ile Leu Pro Ala Val Ile Pro Ile Leu Gly Asp Thr Glu LysGlu Ala 285 290 295 cgc gag cgg cag gaa gaa gtg aat cag cta gtg ata ccagaa gct ggt 3966 Arg Glu Arg Gln Glu Glu Val Asn Gln Leu Val Ile Pro GluAla Gly 300 305 310 ctc tct acc ctg tca agc cat tgc gga gtg gat ttt tcccgc tat cct 4014 Leu Ser Thr Leu Ser Ser His Cys Gly Val Asp Phe Ser ArgTyr Pro 315 320 325 ttg gat gct cca att cgt gag gtg ctg gat gcg gtc ggtgag gtg ggt 4062 Leu Asp Ala Pro Ile Arg Glu Val Leu Asp Ala Val Gly GluVal Gly 330 335 340 ggg acg aga ggt ctt tta gag atg gtg gtg aaa ctg acagag aca gaa 4110 Gly Thr Arg Gly Leu Leu Glu Met Val Val Lys Leu Thr GluThr Glu 345 350 355 360 aac tta acg ttg cgc gac cta ggg gtt cgc tat ggctgg gta ctc gta 4158 Asn Leu Thr Leu Arg Asp Leu Gly Val Arg Tyr Gly TrpVal Leu Val 365 370 375 ccg cag ttg gtt gga acc ccg gag cag gtg gca ggggag ttg gaa tct 4206 Pro Gln Leu Val Gly Thr Pro Glu Gln Val Ala Gly GluLeu Glu Ser 380 385 390 ctg ttc aat gaa ccg gcg gcc gac ggc ttc gtg atctct ccc tac tat 4254 Leu Phe Asn Glu Pro Ala Ala Asp Gly Phe Val Ile SerPro Tyr Tyr 395 400 405 ctg ccc ggc gct tac gag gaa ttt gtc gac aaa gtggtt cct att ttg 4302 Leu Pro Gly Ala Tyr Glu Glu Phe Val Asp Lys Val ValPro Ile Leu 410 415 420 cag gac cgg ggt ctt ttc aga cgg gag tat gaa ggggat acc ttg cgc 4350 Gln Asp Arg Gly Leu Phe Arg Arg Glu Tyr Glu Gly AspThr Leu Arg 425 430 435 440 cag cat ctc ggt ctg gaa gac gtt agc gaa gccgaa gaa gct gta cag 4398 Gln His Leu Gly Leu Glu Asp Val Ser Glu Ala GluGlu Ala Val Gln 445 450 455 ggg gtg agc gaa tgagcacgct ctcagccattggcccgaccc gcgttgcgta 4450 Gly Val Ser Glu 460 tagtaattgt ccggttgcaaacgctttgct cgtggcctca cggacgggga agctagagcg 4510 tcaaggtgtt cttctctcgcagatcgcctt tgcccaaggg gcgacacatt ttgcgtatga 4570 tcatgcagcc tacacccgatttggcggcga gataccaccg ctggtgagcg aagggctgcg 4630 tgctccgggg cggacacgtttgttgggaat cacggttctg aagcctcgcc aagggtttta 4690 tgtgcattct gccggtaagattgcttcacc atcggatctt agagggcgcc gcatcggcct 4750 gagccgagct gcacagaggatccttttcgg ccatctgggc gaggaatatc ggaaccttgg 4810 cccttgggag caaacgctcgtcgccctggg atcgtgggaa gttcgagcgc tcaagcatac 4870 gttggcggcc ggcggtttgagactgaatga cgtcattgtt gaagatgttg aaaacccatg 4930 ggtggatgtc ccgcgacctaaactggatga cagtagggac ttcagctccc gagagttgtt 4990 tgctacggcg gttgaatggcagagtcaaca gttgaaaagc gggcaggtag acgccctgtt 5050 ttcctggctt ccctatgctgccgagcttga acttcaaggt gtggctaagc cggtctttgc 5110 gttgacagga gaggagaatgcctgggcgag cgtttggacg gtcagcgcgg ctctagtgga 5170 gcgcaggccg gagatcgtccaacgcttggt cgactccgtc gtggaggctg cgtcctgggc 5230 aaccgatcac gccaaggagaccattgaaat ccatgccttg aaccttgggg tttccgtgaa 5290 ggccgtggag acgggatttggcgaagggtt tcatagggac ctgcgaccgc ggctggatca 5350 ggcggctctg cgcattctggagcagaccca gcaatttctt ttcgaccacg ggctgatcga 5410 ccggttggtg gatatagagcgttgggcggc ccccgaattt ctggacaacg catctttgtg 5470 aggaggagtt tttctaatgagaacaatcca tgccaattca tctgcagtcc gtgaagatca 5530 tcgtgcttta gacgtggcgacagaactggc caagacgttt cgtgtgaccg ttcgggaaag 5590 ggagcgtgcg gggggaaccccgaaggcgga gcgcgacgcg attcgccgta gtggcctcct 5650 tactctactt atcagtaaagagcgcggggg actcggagaa agttggccga ccgtatacga 5710 agccatcgct gagattgccagcgccgacgc ctcccttggg cacctgtttg gttatcattt 5770 ttcaaatttt gcctatgtggatctctttgc ttcacctgag cagaaggctc gttggtatcc 5830 acaggctgtc cgcgagcgttggttccttgg gaatgcatcc agcgaaaaca atgcgcacgt 5890 tctggattgg cgtgtgacggcgaccccgtt accggacggc agttatgaga tcaacgggac 5950 caaggccttt tgcagcggctcggccgatgc ggacaggttg cttgtgtttg ccgtcaccag 6010 cagggatcca aacggagatggcaggatcgt cgcggcactc atcccctcgg atcgtgctgg 6070 ggttcaggta aatggcgattgggacagcct gggtatgcgt caaaccgata gtgggagcgt 6130 tacattttcg ggtgtggtggtctatcccga cgagttgctg gggacacccg gccaagtgac 6190 ggatgcgttt gcttccggttcgaagcccag tctttggaca cccatcaccc aactgatctt 6250 tacccacctg tacctcggcattgcccgtgg cgctcttgaa gaggccgctc actactcgag 6310 gtcccattcg agaccatttacactcgcagg ggtggagaaa gccaccgagg atccttatgt 6370 gctagcgatt tatggggaatttgctgcaca acttcaggtc gcggaggctg gagcccgaga 6430 ggtggcgttg cgggttcaggaattgtggga gcggaatcac gtcactcctg agcagcgggg 6490 gcagttaatg gtacaagtggccagtgccaa aatcgtcgcc acgcgtttgg tgatcgaact 6550 gacaagccgt ctatatgaagcgatgggggc acgggctgca gcgagccgcc aattcggctt 6610 tgaccgcttt tggcgcgacgcgcgcacgca taccttacat gacccggtag cctataagat 6670 acgcgaagta ggaaactggttcctcaatca ccggtttcca acccccagct tttactcttg 6730 aaatttagtg tgaatagatttatttgagga tgggattggg ggtaacgccg gatgagatcg 6790 acattccagt tccacaaaatgtatctccaa cagatcggcc agcaacaccc ccgtcgcatc 6850 ctcgcgcaga tggaacgtgctgtgactctc aagcattttc gcccagtagt aaagggtccg 6910 cttctcgatg tcccaacggttccacgtcga acaacagggg atggccggaa tcttcaaaca 6970 ccacgttgag aaaatggaccaggaccgaag cctctcggtt ccatcatacc ccgggccgga 7030 caggttcact ctagtgccggataaataccg aagggctgcc ccttggatgt gaggcagccc 7090 gaaaaacatt ttccctgacgggagttttca tcggcgtttc tcttatctcc gcccgagcag 7150 ttcgtcgcgg gtattcacccggcggctcaa taattggtgc gggcggcgca ggcggtttgt 7210 ctccacttca tatatatatccgttgatgat ggtgtccttc ggaatcagcg ggtggttgcg 7270 caggtattcg acttgggccacggtcgcctc gtccacattg tcaaaggtac ggaaccattt 7330 ttcgaaagct gccggctcgctcagtaccag ctcggggagg gagggatcca acggaacccg 7390 ttccacgtct atgttgagtttggcccggag accgtcgaca acttcccggc cgccggcggt 7450 catcatgccg cattcggtgtgattgatcac gatgatttct ttcgtcccga agaagttcag 7510 ggtgagggcc gccgagcggatgacgtcgtc ggtcacaacc cctccggcat tgcggaacac 7570 atgggcatcc ccgggctgcagcccgagaat gtcttccacc ggaagtcgtt catccatgca 7630 ggccaggaca aacagccgcaggttattggg aatccccttc tgcctccgga gcacccattc 7690 ctcatgattt cggatcgcttcgtcaattcg ctcgctcaaa ctcatgatag ttccccctgt 7750 caagcgtctg tgaaaatgtcaggttaactg ttctatgaaa atgtcaggga tgatagttga 7810 ttaaacagcc gccgtcctcttgcagactag ccggatgctg tgctacgctg taactgcttg 7870 ctggagaatg gttttctccagggatggttt gcagcgggct tgcgggggga cgcaggcgcc 7930 gcttcttttt tggccgttgttggcgccggg gtctgtgtgg cctgtgtctc cacacaaggc 7990 caggcccgcc cttgatcccacagccacact tgtccatcca tgccgacacg cacttcgacg 8050 acgctcttcg cttcccagcgcggaacaccg gggacgggct ttggcatgta gcatttccct 8110 ttccagaaga acgtctgcccgccgctgatg cgccggtatt cccgacgcgt gaagatatgc 8170 tccaaaggcg tttcgggcagcggccggtag gccggttcag cttcttgcgg cgcgacggca 8230 aactgacgat tgtgcttggcgataagttcc ggtaacacgc gattggcttc ctccatcgtg 8290 cacacgttgc gcagcctaagttcgatcacc aggcgatcct gaaaggtttg ccagagccgt 8350 tcgatccgtc ctttggcttggggtgacagc gcctcgatat gggtaatgcc cagatcggcg 8410 agggcctgtc cgaaggtggaaagcgacggc ggctcaccgg ccaattcctg ctcgagggtt 8470 ggcttgccct tgggcgggtgaaaaatggag tgttggtcgc tgtagagcgc aagcggtacg 8530 cctttgcgcc taagtccctcgatcatgacg gtcacgtagc cctccagtgt ttcggtcggg 8590 cggaaggtgg ccgcgaccacttccccggtg gcgtcatcga tgatgccgtg cagggtgagc 8650 atgggaccgc gatcctccagccaggcatag ggagaagcat cgatctgcca cagcatgccc 8710 gcctgaggtt tgcggggccggggtcggtga gccttcggac gacggcgcag ccgcgcggga 8770 cgcaacccgc cttccagcagaatgcggcgg accgaagaga cgcttaaatg gatgttttcg 8830 tgttcggcca acagctcggcaaagtgggtg gcattgcttc cgaagtagcg ctcctgatac 8890 aggagcataa cgcgttgtttgagcgaatcg gtcaaggtgt gagccggctt acggccccga 8950 ttcccatgtg cgatcgcttgtgcacctccg tgacgatatt tggccttgag ccgatacgct 9010 tgacggacac tgatgcccaggttgcgtgca acatcctgtt ccgtgagatg gccgtcgatc 9070 catttttcaa tgaccataacgcgtttcagt tcgttctttg tcaaggtgat ctgctccttg 9130 ctcatactga cattttctcggatcagttac accctgacaa tatcacagaa caacaacaac 9190 aatggctggg taatattgacgatttttttt gcaaatgata cattaatagt attacaagct 9250 gttgtgattt tctttgtcgttattaattcg acaaagaagg ggaatgtcgg tacgcttcaa 9310 ccgacgtata aataatgggctttatttagc cgtggagaca ataggacacc taatttggtg 9370 tctttttgtg tttccgcggtttttttatgc ccaaaaaagg aggtaatcga tattggcttc 9430 aaatcgtgaa gaagtgcggagcgcggaaca gtatgtgttg gcggagctgc cccaagaatt 9490 gctcgatatt cgctcttatgatgagtacca catcaatttt tcgggcgggg cagacagctt 9550 ggccgtagcc attttgatgaaatacggcta taaagtgccg ccggagaagc ttatcgatac 9610 cgtcgacctc gagggggggcccggtaccca gcttttgttc cctttagtga gggttaattg 9670 cgcgcttggc gtaatcatggtcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 9730 ttccacacaa catacgagccgggagcataa agtgtaaagc ctggg 9775 2 460 PRT Paenibacillus sp. 2 Met GlyGly Val Gln Ser Met Arg Gln Met His Leu Ala Gly Phe Phe 1 5 10 15 AlaAla Gly Asn Val Thr His His His Gly Ala Trp Arg His Pro Lys 20 25 30 ThrAsp Asn Gly Phe Leu Ser Ile Ser Trp Tyr Gln His Ile Ala Arg 35 40 45 ThrLeu Glu Arg Gly Arg Phe Asp Leu Leu Phe Leu Pro Asp Gly Leu 50 55 60 AlaIle Trp Asp Ser Tyr Gly Asn Asn Leu Asp Ala Gly Leu Arg Phe 65 70 75 80Gly Gly Gln Gly Ala Ala Phe Leu Asp Pro Val Pro Val Leu Ala Thr 85 90 95Met Ala Ala Ala Thr Glu Arg Leu Gly Leu Gly Ala Thr Ile Ser Thr 100 105110 Thr Tyr Tyr Pro Pro Tyr His Val Ala Arg Val Phe Ala Thr Leu Asp 115120 125 His Leu Thr Lys Gly Arg Ala Ala Trp Asn Val Val Thr Ser Leu Asn130 135 140 Asn Ala Glu Ala Arg Asn Phe Gly Tyr Glu Glu His Leu Asp HisAsp 145 150 155 160 Ser Arg Tyr Asp Arg Ala Asp Glu Phe Leu Glu Ile ThrAsp Lys Leu 165 170 175 Trp Arg Ser Trp Asp Gln Asp Ala Leu Leu Leu AspLys Lys Gln Gly 180 185 190 Leu Phe Ala Asp Pro Arg Lys Val His Tyr IleAsp His Ser Gly Thr 195 200 205 Trp Phe Ser Val Arg Gly Pro Leu Gln ValPro Arg Ser Pro Gln Gly 210 215 220 Arg Pro Val Ile Ile Gln Ala Gly SerSer Ala Arg Gly Lys Thr Phe 225 230 235 240 Ala Ala Arg Trp Ala Glu AlaVal Phe Thr Ile Ala Pro Asn Arg Val 245 250 255 Ala Met Arg Ala Phe TyrGlu Asp Leu Lys Lys Gln Val Ile Ala Ala 260 265 270 Gly Arg Arg Pro GluAsn Cys Lys Ile Leu Pro Ala Val Ile Pro Ile 275 280 285 Leu Gly Asp ThrGlu Lys Glu Ala Arg Glu Arg Gln Glu Glu Val Asn 290 295 300 Gln Leu ValIle Pro Glu Ala Gly Leu Ser Thr Leu Ser Ser His Cys 305 310 315 320 GlyVal Asp Phe Ser Arg Tyr Pro Leu Asp Ala Pro Ile Arg Glu Val 325 330 335Leu Asp Ala Val Gly Glu Val Gly Gly Thr Arg Gly Leu Leu Glu Met 340 345350 Val Val Lys Leu Thr Glu Thr Glu Asn Leu Thr Leu Arg Asp Leu Gly 355360 365 Val Arg Tyr Gly Trp Val Leu Val Pro Gln Leu Val Gly Thr Pro Glu370 375 380 Gln Val Ala Gly Glu Leu Glu Ser Leu Phe Asn Glu Pro Ala AlaAsp 385 390 395 400 Gly Phe Val Ile Ser Pro Tyr Tyr Leu Pro Gly Ala TyrGlu Glu Phe 405 410 415 Val Asp Lys Val Val Pro Ile Leu Gln Asp Arg GlyLeu Phe Arg Arg 420 425 430 Glu Tyr Glu Gly Asp Thr Leu Arg Gln His LeuGly Leu Glu Asp Val 435 440 445 Ser Glu Ala Glu Glu Ala Val Gln Gly ValSer Glu 450 455 460 3 9775 DNA Paenibacillus sp. CDS (4410)...(5468) 3gcggccgcgt catcttgccg ccgctcgatg cggtttatcc gatcaatgca aaggacgcaa 60ttcctccttc gcattcctgc ggggtcgaac cgtatcagcc gcaacggatg atttccaatg 120aaatggccgc gatgctgatt tcgaccgtcg tgaatgagct gttttcgtcg aacgccattc 180tcgtccatta tgtcaatttt aatgcaaaga ccgggaactg caggccggtt tatgcagaag 240atgtggccgg cgccaataac gattccgctt cggtagcagc tgcgccgtat gaccaggaag 300ctgactccgg actgcaatca agcgagagtg gccaactcca acatgatccg gacaatgctg 360tatccccgtc tacaaaagag gaggacgctg aaatcctttc tgccgaggag cttcctgcgg 420aacagggggg cgccgaggta gaggtcccgg aaagtggagt ggccggcgtt cgggagaatg 480gtatcagggt aattcgcatc gaaccacttg acgagaaaca cgagaagacg caacacggat 540acggggtacc tgtgctttat catctggaag acgggtccac gctccgtaag ttaattacgg 600ggactcgact gagggacgct aaagcccgtg ttgaaaggct cagtcgcgat cctggcgacc 660ggtggattga acgcaccgaa aacggactcg tgattgaaaa atcgtcgatc ggtcttgtcg 720ggtaaggaaa attgggggcg tattttatgc ccctttttct ttttttataa gggtggaaat 780atcgcgcaag ttaaggggga gcttgagcaa atgaaggtgg ataccgcaaa aattttcaag 840aagtttaaga aggtcattga tacccgcgac atcaatcaca tggacaagca gctttacaat 900tatttgcatc ttcatgcagg cttcatcgcg cattatgaca tctatggctt caaagagaca 960tattccgata aagggtttct tgatttcatt gagcattttg agcagtgcta ttatttgtgc 1020tacggtgaat acggagagtt taaccgcgaa ctgaaggaat atgtgctgca acatgcggag 1080cagatccgcg ctgaatttgc ttataaggcg cagcaacatg aattgaaact gctccagaag 1140ctggcggcaa agcacggcaa aatcatttcc gacgttgcga tgaaccaaga tcaagacatg 1200acggctgctg tggtaccgat gtcgcttgcc gcgaacgggc aattggaatt tgcgctgtga 1260taaatgggaa gggtggagca ttccactctt cctatttatc ttttcaaatt tcggcagcat 1320accacaattt tagagttttg gttggacaat ggctgggtaa tatgtcaagc gtctgtgaaa 1380atgtcaggtt aactgttcta tgaaaatgtc agggatgata gttgattaaa cagccgccgt 1440cctcttgcag actagccgga tgctgtgcta cgctgtaact gcttgctgga gaatggtttt 1500ctccagggat ggtttgcagc gggcttgcgg ggggacgcag gcgccgcttc ttttttggcc 1560gttgttggcg ccggggtctg tgtggcctgt gtctccacac aaggccaggc ccgcccttga 1620tcccacagcc acacttgtcc atccatgccg acacgcactt cgacgacgct cttcgcttcc 1680cagcgcggaa caccggggac gggctttggc atgtagcatt tccctttcca gaagaacgtc 1740tgcccgccgc tgatgcgccg gtattcccga cgcgtgaaga tatgctccaa aggcgtttcg 1800ggcagcggcc ggtaggccgg ttcagcttct tgcggcgcga cggcaaactg acgattgtgc 1860ttggcgataa gttccggtaa cacgcgattg gcttcctcca tcgtgcacac gttgcgcagc 1920ctaagttcga tcaccaggcg atcctgaaag gtttgccaga gccgttcgat ccgtcctttg 1980gcttggggtg acagcgcctc gatatgggta atgcccagat cggcgagggc ctgtccgaag 2040gtggaaagcg acggcggctc accggccaat tcctgctcga gggttggctt gcccttgggc 2100gggtgaaaaa tggagtgttg gtcgctgtag agcgcaagcg gtacgccttt gcgcctaagt 2160ccctcgatca tgacggtcac gtagccctcc agtgtttcgg tcgggcggaa ggtggccgcg 2220accacttccc cggtggcgtc atcgatgatg ccgtgcaggg tgagcatggg accgcgatcc 2280tccagccagg catagggaga agcatcgatc tgccacagca tgcccgcctg aggtttgcgg 2340ggccggggtc ggtgagcctt cggacgacgg cgcagccgcg cgggacgcaa cccgccttcc 2400agcagaatgc ggcggaccga agagacgctt aaatggatgt tttcgtgttc ggccaacagc 2460tcggcaaagt gggtggcatt gcttccgaag tagcgctcct gatacaggag cataacgcgt 2520tgtttgagcg aatcggtcaa ggtgtgagcc ggcttacggc cccgattccc atgtgcgatc 2580gcttgtgcac ctccgtgacg atatttggcc ttgagccgat acgcttgacg gacactgatg 2640cccaggttgc gtgcaacatc ctgttccgtg agatggccgt cgatccattt ttcaatgacc 2700ataacgcgtt tcagttcgtt ctttgtcaag gtgatctgct ccttgctcat actgacattt 2760tctcggatca gttacaccct gacaatatca cagaacaaca acatgagtga ttgcgacggg 2820ttgacaaaat gaatcctgaa cggtatactc cgattcataa atactaatca atttaatcgg 2880gtttacctcg gctgactgga ccaccagagg ccctctgact ttgcggtaat tttgccggaa 2940agcggggggc tttttctttt gcagaggagg gccgaaaaac agttttctgc tcctggatga 3000ccattgaaga acattcacgc aggaacatac atgggaggtg ttcaatcgat gcgtcaaatg 3060catcttgccg gtttttttgc agcgggtaat gtgacccatc accacggggc atggcgtcac 3120ccgaaaactg ataatggttt tttgtctatt tcttggtatc aacacatcgc ccgtacactc 3180gagcgcggcc gctttgacct gctctttctg cctgacggtt tggctatttg ggatagctac 3240ggaaacaatc ttgatgctgg attgagattt ggaggccaag gagccgcttt tctggatccc 3300gtccccgtgc tcgccaccat ggctgcggcc acggagagac tgggcctggg ggccacgatt 3360tcgacaacct actatcctcc ttaccatgtg gcaagagtgt ttgctacgct ggatcactta 3420acaaaaggaa gggcagcctg gaatgtcgtg acctcactca acaacgccga ggccaggaac 3480tttgggtatg aggaacacct ggatcacgat agtcggtacg accgtgccga tgagtttctt 3540gagattacag ataaattgtg gaggagttgg gatcaggatg cattgctcct cgacaaaaaa 3600cagggtcttt ttgctgatcc cagaaaggtc cactatattg atcactccgg aacctggttc 3660tccgtccggg gcccgttaca agtcccgcgg tcgccacagg gtcgtcctgt catcattcag 3720gcgggatcct ccgcccgtgg aaagacattt gctgctcggt gggcagaagc cgttttcacc 3780attgcgccga accgagtcgc gatgcgggcg ttttacgaag acttgaaaaa acaggtaatc 3840gccgcaggac gccgtcccga gaattgcaaa atactccctg ccgtcattcc gattcttggc 3900gatacggaga aggaagcgcg cgagcggcag gaagaagtga atcagctagt gataccagaa 3960gctggtctct ctaccctgtc aagccattgc ggagtggatt tttcccgcta tcctttggat 4020gctccaattc gtgaggtgct ggatgcggtc ggtgaggtgg gtgggacgag aggtctttta 4080gagatggtgg tgaaactgac agagacagaa aacttaacgt tgcgcgacct aggggttcgc 4140tatggctggg tactcgtacc gcagttggtt ggaaccccgg agcaggtggc aggggagttg 4200gaatctctgt tcaatgaacc ggcggccgac ggcttcgtga tctctcccta ctatctgccc 4260ggcgcttacg aggaatttgt cgacaaagtg gttcctattt tgcaggaccg gggtcttttc 4320agacgggagt atgaagggga taccttgcgc cagcatctcg gtctggaaga cgttagcgaa 4380gccgaagaag ctgtacaggg ggtgagcga atg agc acg ctc tca gcc att ggc 4433 MetSer Thr Leu Ser Ala Ile Gly 1 5 ccg acc cgc gtt gcg tat agt aat tgt ccggtt gca aac gct ttg ctc 4481 Pro Thr Arg Val Ala Tyr Ser Asn Cys Pro ValAla Asn Ala Leu Leu 10 15 20 gtg gcc tca cgg acg ggg aag cta gag cgt caaggt gtt ctt ctc tcg 4529 Val Ala Ser Arg Thr Gly Lys Leu Glu Arg Gln GlyVal Leu Leu Ser 25 30 35 40 cag atc gcc ttt gcc caa ggg gcg aca cat tttgcg tat gat cat gca 4577 Gln Ile Ala Phe Ala Gln Gly Ala Thr His Phe AlaTyr Asp His Ala 45 50 55 gcc tac acc cga ttt ggc ggc gag ata cca ccg ctggtg agc gaa ggg 4625 Ala Tyr Thr Arg Phe Gly Gly Glu Ile Pro Pro Leu ValSer Glu Gly 60 65 70 ctg cgt gct ccg ggg cgg aca cgt ttg ttg gga atc acggtt ctg aag 4673 Leu Arg Ala Pro Gly Arg Thr Arg Leu Leu Gly Ile Thr ValLeu Lys 75 80 85 cct cgc caa ggg ttt tat gtg cat tct gcc ggt aag att gcttca cca 4721 Pro Arg Gln Gly Phe Tyr Val His Ser Ala Gly Lys Ile Ala SerPro 90 95 100 tcg gat ctt aga ggg cgc cgc atc ggc ctg agc cga gct gcacag agg 4769 Ser Asp Leu Arg Gly Arg Arg Ile Gly Leu Ser Arg Ala Ala GlnArg 105 110 115 120 atc ctt ttc ggc cat ctg ggc gag gaa tat cgg aac cttggc cct tgg 4817 Ile Leu Phe Gly His Leu Gly Glu Glu Tyr Arg Asn Leu GlyPro Trp 125 130 135 gag caa acg ctc gtc gcc ctg gga tcg tgg gaa gtt cgagcg ctc aag 4865 Glu Gln Thr Leu Val Ala Leu Gly Ser Trp Glu Val Arg AlaLeu Lys 140 145 150 cat acg ttg gcg gcc ggc ggt ttg aga ctg aat gac gtcatt gtt gaa 4913 His Thr Leu Ala Ala Gly Gly Leu Arg Leu Asn Asp Val IleVal Glu 155 160 165 gat gtt gaa aac cca tgg gtg gat gtc ccg cga cct aaactg gat gac 4961 Asp Val Glu Asn Pro Trp Val Asp Val Pro Arg Pro Lys LeuAsp Asp 170 175 180 agt agg gac ttc agc tcc cga gag ttg ttt gct acg gcggtt gaa tgg 5009 Ser Arg Asp Phe Ser Ser Arg Glu Leu Phe Ala Thr Ala ValGlu Trp 185 190 195 200 cag agt caa cag ttg aaa agc ggg cag gta gac gccctg ttt tcc tgg 5057 Gln Ser Gln Gln Leu Lys Ser Gly Gln Val Asp Ala LeuPhe Ser Trp 205 210 215 ctt ccc tat gct gcc gag ctt gaa ctt caa ggt gtggct aag ccg gtc 5105 Leu Pro Tyr Ala Ala Glu Leu Glu Leu Gln Gly Val AlaLys Pro Val 220 225 230 ttt gcg ttg aca gga gag gag aat gcc tgg gcg agcgtt tgg acg gtc 5153 Phe Ala Leu Thr Gly Glu Glu Asn Ala Trp Ala Ser ValTrp Thr Val 235 240 245 agc gcg gct cta gtg gag cgc agg ccg gag atc gtccaa cgc ttg gtc 5201 Ser Ala Ala Leu Val Glu Arg Arg Pro Glu Ile Val GlnArg Leu Val 250 255 260 gac tcc gtc gtg gag gct gcg tcc tgg gca acc gatcac gcc aag gag 5249 Asp Ser Val Val Glu Ala Ala Ser Trp Ala Thr Asp HisAla Lys Glu 265 270 275 280 acc att gaa atc cat gcc ttg aac ctt ggg gtttcc gtg aag gcc gtg 5297 Thr Ile Glu Ile His Ala Leu Asn Leu Gly Val SerVal Lys Ala Val 285 290 295 gag acg gga ttt ggc gaa ggg ttt cat agg gacctg cga ccg cgg ctg 5345 Glu Thr Gly Phe Gly Glu Gly Phe His Arg Asp LeuArg Pro Arg Leu 300 305 310 gat cag gcg gct ctg cgc att ctg gag cag acccag caa ttt ctt ttc 5393 Asp Gln Ala Ala Leu Arg Ile Leu Glu Gln Thr GlnGln Phe Leu Phe 315 320 325 gac cac ggg ctg atc gac cgg ttg gtg gat atagag cgt tgg gcg gcc 5441 Asp His Gly Leu Ile Asp Arg Leu Val Asp Ile GluArg Trp Ala Ala 330 335 340 ccc gaa ttt ctg gac aac gca tct ttgtgaggaggag tttttctaat 5488 Pro Glu Phe Leu Asp Asn Ala Ser Leu 345 350gagaacaatc catgccaatt catctgcagt ccgtgaagat catcgtgctt tagacgtggc 5548gacagaactg gccaagacgt ttcgtgtgac cgttcgggaa agggagcgtg cggggggaac 5608cccgaaggcg gagcgcgacg cgattcgccg tagtggcctc cttactctac ttatcagtaa 5668agagcgcggg ggactcggag aaagttggcc gaccgtatac gaagccatcg ctgagattgc 5728cagcgccgac gcctcccttg ggcacctgtt tggttatcat ttttcaaatt ttgcctatgt 5788ggatctcttt gcttcacctg agcagaaggc tcgttggtat ccacaggctg tccgcgagcg 5848ttggttcctt gggaatgcat ccagcgaaaa caatgcgcac gttctggatt ggcgtgtgac 5908ggcgaccccg ttaccggacg gcagttatga gatcaacggg accaaggcct tttgcagcgg 5968ctcggccgat gcggacaggt tgcttgtgtt tgccgtcacc agcagggatc caaacggaga 6028tggcaggatc gtcgcggcac tcatcccctc ggatcgtgct ggggttcagg taaatggcga 6088ttgggacagc ctgggtatgc gtcaaaccga tagtgggagc gttacatttt cgggtgtggt 6148ggtctatccc gacgagttgc tggggacacc cggccaagtg acggatgcgt ttgcttccgg 6208ttcgaagccc agtctttgga cacccatcac ccaactgatc tttacccacc tgtacctcgg 6268cattgcccgt ggcgctcttg aagaggccgc tcactactcg aggtcccatt cgagaccatt 6328tacactcgca ggggtggaga aagccaccga ggatccttat gtgctagcga tttatgggga 6388atttgctgca caacttcagg tcgcggaggc tggagcccga gaggtggcgt tgcgggttca 6448ggaattgtgg gagcggaatc acgtcactcc tgagcagcgg gggcagttaa tggtacaagt 6508ggccagtgcc aaaatcgtcg ccacgcgttt ggtgatcgaa ctgacaagcc gtctatatga 6568agcgatgggg gcacgggctg cagcgagccg ccaattcggc tttgaccgct tttggcgcga 6628cgcgcgcacg cataccttac atgacccggt agcctataag atacgcgaag taggaaactg 6688gttcctcaat caccggtttc caacccccag cttttactct tgaaatttag tgtgaataga 6748tttatttgag gatgggattg ggggtaacgc cggatgagat cgacattcca gttccacaaa 6808atgtatctcc aacagatcgg ccagcaacac ccccgtcgca tcctcgcgca gatggaacgt 6868gctgtgactc tcaagcattt tcgcccagta gtaaagggtc cgcttctcga tgtcccaacg 6928gttccacgtc gaacaacagg ggatggccgg aatcttcaaa caccacgttg agaaaatgga 6988ccaggaccga agcctctcgg ttccatcata ccccgggccg gacaggttca ctctagtgcc 7048ggataaatac cgaagggctg ccccttggat gtgaggcagc ccgaaaaaca ttttccctga 7108cgggagtttt catcggcgtt tctcttatct ccgcccgagc agttcgtcgc gggtattcac 7168ccggcggctc aataattggt gcgggcggcg caggcggttt gtctccactt catatatata 7228tccgttgatg atggtgtcct tcggaatcag cgggtggttg cgcaggtatt cgacttgggc 7288cacggtcgcc tcgtccacat tgtcaaaggt acggaaccat ttttcgaaag ctgccggctc 7348gctcagtacc agctcgggga gggagggatc caacggaacc cgttccacgt ctatgttgag 7408tttggcccgg agaccgtcga caacttcccg gccgccggcg gtcatcatgc cgcattcggt 7468gtgattgatc acgatgattt ctttcgtccc gaagaagttc agggtgaggg ccgccgagcg 7528gatgacgtcg tcggtcacaa cccctccggc attgcggaac acatgggcat ccccgggctg 7588cagcccgaga atgtcttcca ccggaagtcg ttcatccatg caggccagga caaacagccg 7648caggttattg ggaatcccct tctgcctccg gagcacccat tcctcatgat ttcggatcgc 7708ttcgtcaatt cgctcgctca aactcatgat agttccccct gtcaagcgtc tgtgaaaatg 7768tcaggttaac tgttctatga aaatgtcagg gatgatagtt gattaaacag ccgccgtcct 7828cttgcagact agccggatgc tgtgctacgc tgtaactgct tgctggagaa tggttttctc 7888cagggatggt ttgcagcggg cttgcggggg gacgcaggcg ccgcttcttt tttggccgtt 7948gttggcgccg gggtctgtgt ggcctgtgtc tccacacaag gccaggcccg cccttgatcc 8008cacagccaca cttgtccatc catgccgaca cgcacttcga cgacgctctt cgcttcccag 8068cgcggaacac cggggacggg ctttggcatg tagcatttcc ctttccagaa gaacgtctgc 8128ccgccgctga tgcgccggta ttcccgacgc gtgaagatat gctccaaagg cgtttcgggc 8188agcggccggt aggccggttc agcttcttgc ggcgcgacgg caaactgacg attgtgcttg 8248gcgataagtt ccggtaacac gcgattggct tcctccatcg tgcacacgtt gcgcagccta 8308agttcgatca ccaggcgatc ctgaaaggtt tgccagagcc gttcgatccg tcctttggct 8368tggggtgaca gcgcctcgat atgggtaatg cccagatcgg cgagggcctg tccgaaggtg 8428gaaagcgacg gcggctcacc ggccaattcc tgctcgaggg ttggcttgcc cttgggcggg 8488tgaaaaatgg agtgttggtc gctgtagagc gcaagcggta cgcctttgcg cctaagtccc 8548tcgatcatga cggtcacgta gccctccagt gtttcggtcg ggcggaaggt ggccgcgacc 8608acttccccgg tggcgtcatc gatgatgccg tgcagggtga gcatgggacc gcgatcctcc 8668agccaggcat agggagaagc atcgatctgc cacagcatgc ccgcctgagg tttgcggggc 8728cggggtcggt gagccttcgg acgacggcgc agccgcgcgg gacgcaaccc gccttccagc 8788agaatgcggc ggaccgaaga gacgcttaaa tggatgtttt cgtgttcggc caacagctcg 8848gcaaagtggg tggcattgct tccgaagtag cgctcctgat acaggagcat aacgcgttgt 8908ttgagcgaat cggtcaaggt gtgagccggc ttacggcccc gattcccatg tgcgatcgct 8968tgtgcacctc cgtgacgata tttggccttg agccgatacg cttgacggac actgatgccc 9028aggttgcgtg caacatcctg ttccgtgaga tggccgtcga tccatttttc aatgaccata 9088acgcgtttca gttcgttctt tgtcaaggtg atctgctcct tgctcatact gacattttct 9148cggatcagtt acaccctgac aatatcacag aacaacaaca acaatggctg ggtaatattg 9208acgatttttt ttgcaaatga tacattaata gtattacaag ctgttgtgat tttctttgtc 9268gttattaatt cgacaaagaa ggggaatgtc ggtacgcttc aaccgacgta taaataatgg 9328gctttattta gccgtggaga caataggaca cctaatttgg tgtctttttg tgtttccgcg 9388gtttttttat gcccaaaaaa ggaggtaatc gatattggct tcaaatcgtg aagaagtgcg 9448gagcgcggaa cagtatgtgt tggcggagct gccccaagaa ttgctcgata ttcgctctta 9508tgatgagtac cacatcaatt tttcgggcgg ggcagacagc ttggccgtag ccattttgat 9568gaaatacggc tataaagtgc cgccggagaa gcttatcgat accgtcgacc tcgagggggg 9628gcccggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat 9688ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 9748ccgggagcat aaagtgtaaa gcctggg 9775 4 353 PRT Paenibacillus sp. 4 Met SerThr Leu Ser Ala Ile Gly Pro Thr Arg Val Ala Tyr Ser Asn 1 5 10 15 CysPro Val Ala Asn Ala Leu Leu Val Ala Ser Arg Thr Gly Lys Leu 20 25 30 GluArg Gln Gly Val Leu Leu Ser Gln Ile Ala Phe Ala Gln Gly Ala 35 40 45 ThrHis Phe Ala Tyr Asp His Ala Ala Tyr Thr Arg Phe Gly Gly Glu 50 55 60 IlePro Pro Leu Val Ser Glu Gly Leu Arg Ala Pro Gly Arg Thr Arg 65 70 75 80Leu Leu Gly Ile Thr Val Leu Lys Pro Arg Gln Gly Phe Tyr Val His 85 90 95Ser Ala Gly Lys Ile Ala Ser Pro Ser Asp Leu Arg Gly Arg Arg Ile 100 105110 Gly Leu Ser Arg Ala Ala Gln Arg Ile Leu Phe Gly His Leu Gly Glu 115120 125 Glu Tyr Arg Asn Leu Gly Pro Trp Glu Gln Thr Leu Val Ala Leu Gly130 135 140 Ser Trp Glu Val Arg Ala Leu Lys His Thr Leu Ala Ala Gly GlyLeu 145 150 155 160 Arg Leu Asn Asp Val Ile Val Glu Asp Val Glu Asn ProTrp Val Asp 165 170 175 Val Pro Arg Pro Lys Leu Asp Asp Ser Arg Asp PheSer Ser Arg Glu 180 185 190 Leu Phe Ala Thr Ala Val Glu Trp Gln Ser GlnGln Leu Lys Ser Gly 195 200 205 Gln Val Asp Ala Leu Phe Ser Trp Leu ProTyr Ala Ala Glu Leu Glu 210 215 220 Leu Gln Gly Val Ala Lys Pro Val PheAla Leu Thr Gly Glu Glu Asn 225 230 235 240 Ala Trp Ala Ser Val Trp ThrVal Ser Ala Ala Leu Val Glu Arg Arg 245 250 255 Pro Glu Ile Val Gln ArgLeu Val Asp Ser Val Val Glu Ala Ala Ser 260 265 270 Trp Ala Thr Asp HisAla Lys Glu Thr Ile Glu Ile His Ala Leu Asn 275 280 285 Leu Gly Val SerVal Lys Ala Val Glu Thr Gly Phe Gly Glu Gly Phe 290 295 300 His Arg AspLeu Arg Pro Arg Leu Asp Gln Ala Ala Leu Arg Ile Leu 305 310 315 320 GluGln Thr Gln Gln Phe Leu Phe Asp His Gly Leu Ile Asp Arg Leu 325 330 335Val Asp Ile Glu Arg Trp Ala Ala Pro Glu Phe Leu Asp Asn Ala Ser 340 345350 Leu 5 9775 DNA Paenibacillus sp. CDS (5487)...(6728) 5 gcggccgcgtcatcttgccg ccgctcgatg cggtttatcc gatcaatgca aaggacgcaa 60 ttcctccttcgcattcctgc ggggtcgaac cgtatcagcc gcaacggatg atttccaatg 120 aaatggccgcgatgctgatt tcgaccgtcg tgaatgagct gttttcgtcg aacgccattc 180 tcgtccattatgtcaatttt aatgcaaaga ccgggaactg caggccggtt tatgcagaag 240 atgtggccggcgccaataac gattccgctt cggtagcagc tgcgccgtat gaccaggaag 300 ctgactccggactgcaatca agcgagagtg gccaactcca acatgatccg gacaatgctg 360 tatccccgtctacaaaagag gaggacgctg aaatcctttc tgccgaggag cttcctgcgg 420 aacaggggggcgccgaggta gaggtcccgg aaagtggagt ggccggcgtt cgggagaatg 480 gtatcagggtaattcgcatc gaaccacttg acgagaaaca cgagaagacg caacacggat 540 acggggtacctgtgctttat catctggaag acgggtccac gctccgtaag ttaattacgg 600 ggactcgactgagggacgct aaagcccgtg ttgaaaggct cagtcgcgat cctggcgacc 660 ggtggattgaacgcaccgaa aacggactcg tgattgaaaa atcgtcgatc ggtcttgtcg 720 ggtaaggaaaattgggggcg tattttatgc ccctttttct ttttttataa gggtggaaat 780 atcgcgcaagttaaggggga gcttgagcaa atgaaggtgg ataccgcaaa aattttcaag 840 aagtttaagaaggtcattga tacccgcgac atcaatcaca tggacaagca gctttacaat 900 tatttgcatcttcatgcagg cttcatcgcg cattatgaca tctatggctt caaagagaca 960 tattccgataaagggtttct tgatttcatt gagcattttg agcagtgcta ttatttgtgc 1020 tacggtgaatacggagagtt taaccgcgaa ctgaaggaat atgtgctgca acatgcggag 1080 cagatccgcgctgaatttgc ttataaggcg cagcaacatg aattgaaact gctccagaag 1140 ctggcggcaaagcacggcaa aatcatttcc gacgttgcga tgaaccaaga tcaagacatg 1200 acggctgctgtggtaccgat gtcgcttgcc gcgaacgggc aattggaatt tgcgctgtga 1260 taaatgggaagggtggagca ttccactctt cctatttatc ttttcaaatt tcggcagcat 1320 accacaattttagagttttg gttggacaat ggctgggtaa tatgtcaagc gtctgtgaaa 1380 atgtcaggttaactgttcta tgaaaatgtc agggatgata gttgattaaa cagccgccgt 1440 cctcttgcagactagccgga tgctgtgcta cgctgtaact gcttgctgga gaatggtttt 1500 ctccagggatggtttgcagc gggcttgcgg ggggacgcag gcgccgcttc ttttttggcc 1560 gttgttggcgccggggtctg tgtggcctgt gtctccacac aaggccaggc ccgcccttga 1620 tcccacagccacacttgtcc atccatgccg acacgcactt cgacgacgct cttcgcttcc 1680 cagcgcggaacaccggggac gggctttggc atgtagcatt tccctttcca gaagaacgtc 1740 tgcccgccgctgatgcgccg gtattcccga cgcgtgaaga tatgctccaa aggcgtttcg 1800 ggcagcggccggtaggccgg ttcagcttct tgcggcgcga cggcaaactg acgattgtgc 1860 ttggcgataagttccggtaa cacgcgattg gcttcctcca tcgtgcacac gttgcgcagc 1920 ctaagttcgatcaccaggcg atcctgaaag gtttgccaga gccgttcgat ccgtcctttg 1980 gcttggggtgacagcgcctc gatatgggta atgcccagat cggcgagggc ctgtccgaag 2040 gtggaaagcgacggcggctc accggccaat tcctgctcga gggttggctt gcccttgggc 2100 gggtgaaaaatggagtgttg gtcgctgtag agcgcaagcg gtacgccttt gcgcctaagt 2160 ccctcgatcatgacggtcac gtagccctcc agtgtttcgg tcgggcggaa ggtggccgcg 2220 accacttccccggtggcgtc atcgatgatg ccgtgcaggg tgagcatggg accgcgatcc 2280 tccagccaggcatagggaga agcatcgatc tgccacagca tgcccgcctg aggtttgcgg 2340 ggccggggtcggtgagcctt cggacgacgg cgcagccgcg cgggacgcaa cccgccttcc 2400 agcagaatgcggcggaccga agagacgctt aaatggatgt tttcgtgttc ggccaacagc 2460 tcggcaaagtgggtggcatt gcttccgaag tagcgctcct gatacaggag cataacgcgt 2520 tgtttgagcgaatcggtcaa ggtgtgagcc ggcttacggc cccgattccc atgtgcgatc 2580 gcttgtgcacctccgtgacg atatttggcc ttgagccgat acgcttgacg gacactgatg 2640 cccaggttgcgtgcaacatc ctgttccgtg agatggccgt cgatccattt ttcaatgacc 2700 ataacgcgtttcagttcgtt ctttgtcaag gtgatctgct ccttgctcat actgacattt 2760 tctcggatcagttacaccct gacaatatca cagaacaaca acatgagtga ttgcgacggg 2820 ttgacaaaatgaatcctgaa cggtatactc cgattcataa atactaatca atttaatcgg 2880 gtttacctcggctgactgga ccaccagagg ccctctgact ttgcggtaat tttgccggaa 2940 agcggggggctttttctttt gcagaggagg gccgaaaaac agttttctgc tcctggatga 3000 ccattgaagaacattcacgc aggaacatac atgggaggtg ttcaatcgat gcgtcaaatg 3060 catcttgccggtttttttgc agcgggtaat gtgacccatc accacggggc atggcgtcac 3120 ccgaaaactgataatggttt tttgtctatt tcttggtatc aacacatcgc ccgtacactc 3180 gagcgcggccgctttgacct gctctttctg cctgacggtt tggctatttg ggatagctac 3240 ggaaacaatcttgatgctgg attgagattt ggaggccaag gagccgcttt tctggatccc 3300 gtccccgtgctcgccaccat ggctgcggcc acggagagac tgggcctggg ggccacgatt 3360 tcgacaacctactatcctcc ttaccatgtg gcaagagtgt ttgctacgct ggatcactta 3420 acaaaaggaagggcagcctg gaatgtcgtg acctcactca acaacgccga ggccaggaac 3480 tttgggtatgaggaacacct ggatcacgat agtcggtacg accgtgccga tgagtttctt 3540 gagattacagataaattgtg gaggagttgg gatcaggatg cattgctcct cgacaaaaaa 3600 cagggtctttttgctgatcc cagaaaggtc cactatattg atcactccgg aacctggttc 3660 tccgtccggggcccgttaca agtcccgcgg tcgccacagg gtcgtcctgt catcattcag 3720 gcgggatcctccgcccgtgg aaagacattt gctgctcggt gggcagaagc cgttttcacc 3780 attgcgccgaaccgagtcgc gatgcgggcg ttttacgaag acttgaaaaa acaggtaatc 3840 gccgcaggacgccgtcccga gaattgcaaa atactccctg ccgtcattcc gattcttggc 3900 gatacggagaaggaagcgcg cgagcggcag gaagaagtga atcagctagt gataccagaa 3960 gctggtctctctaccctgtc aagccattgc ggagtggatt tttcccgcta tcctttggat 4020 gctccaattcgtgaggtgct ggatgcggtc ggtgaggtgg gtgggacgag aggtctttta 4080 gagatggtggtgaaactgac agagacagaa aacttaacgt tgcgcgacct aggggttcgc 4140 tatggctgggtactcgtacc gcagttggtt ggaaccccgg agcaggtggc aggggagttg 4200 gaatctctgttcaatgaacc ggcggccgac ggcttcgtga tctctcccta ctatctgccc 4260 ggcgcttacgaggaatttgt cgacaaagtg gttcctattt tgcaggaccg gggtcttttc 4320 agacgggagtatgaagggga taccttgcgc cagcatctcg gtctggaaga cgttagcgaa 4380 gccgaagaagctgtacaggg ggtgagcgaa tgagcacgct ctcagccatt ggcccgaccc 4440 gcgttgcgtatagtaattgt ccggttgcaa acgctttgct cgtggcctca cggacgggga 4500 agctagagcgtcaaggtgtt cttctctcgc agatcgcctt tgcccaaggg gcgacacatt 4560 ttgcgtatgatcatgcagcc tacacccgat ttggcggcga gataccaccg ctggtgagcg 4620 aagggctgcgtgctccgggg cggacacgtt tgttgggaat cacggttctg aagcctcgcc 4680 aagggttttatgtgcattct gccggtaaga ttgcttcacc atcggatctt agagggcgcc 4740 gcatcggcctgagccgagct gcacagagga tccttttcgg ccatctgggc gaggaatatc 4800 ggaaccttggcccttgggag caaacgctcg tcgccctggg atcgtgggaa gttcgagcgc 4860 tcaagcatacgttggcggcc ggcggtttga gactgaatga cgtcattgtt gaagatgttg 4920 aaaacccatgggtggatgtc ccgcgaccta aactggatga cagtagggac ttcagctccc 4980 gagagttgtttgctacggcg gttgaatggc agagtcaaca gttgaaaagc gggcaggtag 5040 acgccctgttttcctggctt ccctatgctg ccgagcttga acttcaaggt gtggctaagc 5100 cggtctttgcgttgacagga gaggagaatg cctgggcgag cgtttggacg gtcagcgcgg 5160 ctctagtggagcgcaggccg gagatcgtcc aacgcttggt cgactccgtc gtggaggctg 5220 cgtcctgggcaaccgatcac gccaaggaga ccattgaaat ccatgccttg aaccttgggg 5280 tttccgtgaaggccgtggag acgggatttg gcgaagggtt tcatagggac ctgcgaccgc 5340 ggctggatcaggcggctctg cgcattctgg agcagaccca gcaatttctt ttcgaccacg 5400 ggctgatcgaccggttggtg gatatagagc gttgggcggc ccccgaattt ctggacaacg 5460 catctttgtgaggaggagtt tttcta atg aga aca atc cat gcc aat tca tct 5513 Met Arg ThrIle His Ala Asn Ser Ser 1 5 gca gtc cgt gaa gat cat cgt gct tta gac gtggcg aca gaa ctg gcc 5561 Ala Val Arg Glu Asp His Arg Ala Leu Asp Val AlaThr Glu Leu Ala 10 15 20 25 aag acg ttt cgt gtg acc gtt cgg gaa agg gagcgt gcg ggg gga acc 5609 Lys Thr Phe Arg Val Thr Val Arg Glu Arg Glu ArgAla Gly Gly Thr 30 35 40 ccg aag gcg gag cgc gac gcg att cgc cgt agt ggcctc ctt act cta 5657 Pro Lys Ala Glu Arg Asp Ala Ile Arg Arg Ser Gly LeuLeu Thr Leu 45 50 55 ctt atc agt aaa gag cgc ggg gga ctc gga gaa agt tggccg acc gta 5705 Leu Ile Ser Lys Glu Arg Gly Gly Leu Gly Glu Ser Trp ProThr Val 60 65 70 tac gaa gcc atc gct gag att gcc agc gcc gac gcc tcc cttggg cac 5753 Tyr Glu Ala Ile Ala Glu Ile Ala Ser Ala Asp Ala Ser Leu GlyHis 75 80 85 ctg ttt ggt tat cat ttt tca aat ttt gcc tat gtg gat ctc tttgct 5801 Leu Phe Gly Tyr His Phe Ser Asn Phe Ala Tyr Val Asp Leu Phe Ala90 95 100 105 tca cct gag cag aag gct cgt tgg tat cca cag gct gtc cgcgag cgt 5849 Ser Pro Glu Gln Lys Ala Arg Trp Tyr Pro Gln Ala Val Arg GluArg 110 115 120 tgg ttc ctt ggg aat gca tcc agc gaa aac aat gcg cac gttctg gat 5897 Trp Phe Leu Gly Asn Ala Ser Ser Glu Asn Asn Ala His Val LeuAsp 125 130 135 tgg cgt gtg acg gcg acc ccg tta ccg gac ggc agt tat gagatc aac 5945 Trp Arg Val Thr Ala Thr Pro Leu Pro Asp Gly Ser Tyr Glu IleAsn 140 145 150 ggg acc aag gcc ttt tgc agc ggc tcg gcc gat gcg gac aggttg ctt 5993 Gly Thr Lys Ala Phe Cys Ser Gly Ser Ala Asp Ala Asp Arg LeuLeu 155 160 165 gtg ttt gcc gtc acc agc agg gat cca aac gga gat ggc aggatc gtc 6041 Val Phe Ala Val Thr Ser Arg Asp Pro Asn Gly Asp Gly Arg IleVal 170 175 180 185 gcg gca ctc atc ccc tcg gat cgt gct ggg gtt cag gtaaat ggc gat 6089 Ala Ala Leu Ile Pro Ser Asp Arg Ala Gly Val Gln Val AsnGly Asp 190 195 200 tgg gac agc ctg ggt atg cgt caa acc gat agt ggg agcgtt aca ttt 6137 Trp Asp Ser Leu Gly Met Arg Gln Thr Asp Ser Gly Ser ValThr Phe 205 210 215 tcg ggt gtg gtg gtc tat ccc gac gag ttg ctg ggg acaccc ggc caa 6185 Ser Gly Val Val Val Tyr Pro Asp Glu Leu Leu Gly Thr ProGly Gln 220 225 230 gtg acg gat gcg ttt gct tcc ggt tcg aag ccc agt ctttgg aca ccc 6233 Val Thr Asp Ala Phe Ala Ser Gly Ser Lys Pro Ser Leu TrpThr Pro 235 240 245 atc acc caa ctg atc ttt acc cac ctg tac ctc ggc attgcc cgt ggc 6281 Ile Thr Gln Leu Ile Phe Thr His Leu Tyr Leu Gly Ile AlaArg Gly 250 255 260 265 gct ctt gaa gag gcc gct cac tac tcg agg tcc cattcg aga cca ttt 6329 Ala Leu Glu Glu Ala Ala His Tyr Ser Arg Ser His SerArg Pro Phe 270 275 280 aca ctc gca ggg gtg gag aaa gcc acc gag gat ccttat gtg cta gcg 6377 Thr Leu Ala Gly Val Glu Lys Ala Thr Glu Asp Pro TyrVal Leu Ala 285 290 295 att tat ggg gaa ttt gct gca caa ctt cag gtc gcggag gct gga gcc 6425 Ile Tyr Gly Glu Phe Ala Ala Gln Leu Gln Val Ala GluAla Gly Ala 300 305 310 cga gag gtg gcg ttg cgg gtt cag gaa ttg tgg gagcgg aat cac gtc 6473 Arg Glu Val Ala Leu Arg Val Gln Glu Leu Trp Glu ArgAsn His Val 315 320 325 act cct gag cag cgg ggg cag tta atg gta caa gtggcc agt gcc aaa 6521 Thr Pro Glu Gln Arg Gly Gln Leu Met Val Gln Val AlaSer Ala Lys 330 335 340 345 atc gtc gcc acg cgt ttg gtg atc gaa ctg acaagc cgt cta tat gaa 6569 Ile Val Ala Thr Arg Leu Val Ile Glu Leu Thr SerArg Leu Tyr Glu 350 355 360 gcg atg ggg gca cgg gct gca gcg agc cgc caattc ggc ttt gac cgc 6617 Ala Met Gly Ala Arg Ala Ala Ala Ser Arg Gln PheGly Phe Asp Arg 365 370 375 ttt tgg cgc gac gcg cgc acg cat acc tta catgac ccg gta gcc tat 6665 Phe Trp Arg Asp Ala Arg Thr His Thr Leu His AspPro Val Ala Tyr 380 385 390 aag ata cgc gaa gta gga aac tgg ttc ctc aatcac cgg ttt cca acc 6713 Lys Ile Arg Glu Val Gly Asn Trp Phe Leu Asn HisArg Phe Pro Thr 395 400 405 ccc agc ttt tac tct tgaaatttag tgtgaatagatttatttgag gatgggattg 6768 Pro Ser Phe Tyr Ser 410 ggggtaacgc cggatgagatcgacattcca gttccacaaa atgtatctcc aacagatcgg 6828 ccagcaacac ccccgtcgcatcctcgcgca gatggaacgt gctgtgactc tcaagcattt 6888 tcgcccagta gtaaagggtccgcttctcga tgtcccaacg gttccacgtc gaacaacagg 6948 ggatggccgg aatcttcaaacaccacgttg agaaaatgga ccaggaccga agcctctcgg 7008 ttccatcata ccccgggccggacaggttca ctctagtgcc ggataaatac cgaagggctg 7068 ccccttggat gtgaggcagcccgaaaaaca ttttccctga cgggagtttt catcggcgtt 7128 tctcttatct ccgcccgagcagttcgtcgc gggtattcac ccggcggctc aataattggt 7188 gcgggcggcg caggcggtttgtctccactt catatatata tccgttgatg atggtgtcct 7248 tcggaatcag cgggtggttgcgcaggtatt cgacttgggc cacggtcgcc tcgtccacat 7308 tgtcaaaggt acggaaccatttttcgaaag ctgccggctc gctcagtacc agctcgggga 7368 gggagggatc caacggaacccgttccacgt ctatgttgag tttggcccgg agaccgtcga 7428 caacttcccg gccgccggcggtcatcatgc cgcattcggt gtgattgatc acgatgattt 7488 ctttcgtccc gaagaagttcagggtgaggg ccgccgagcg gatgacgtcg tcggtcacaa 7548 cccctccggc attgcggaacacatgggcat ccccgggctg cagcccgaga atgtcttcca 7608 ccggaagtcg ttcatccatgcaggccagga caaacagccg caggttattg ggaatcccct 7668 tctgcctccg gagcacccattcctcatgat ttcggatcgc ttcgtcaatt cgctcgctca 7728 aactcatgat agttccccctgtcaagcgtc tgtgaaaatg tcaggttaac tgttctatga 7788 aaatgtcagg gatgatagttgattaaacag ccgccgtcct cttgcagact agccggatgc 7848 tgtgctacgc tgtaactgcttgctggagaa tggttttctc cagggatggt ttgcagcggg 7908 cttgcggggg gacgcaggcgccgcttcttt tttggccgtt gttggcgccg gggtctgtgt 7968 ggcctgtgtc tccacacaaggccaggcccg cccttgatcc cacagccaca cttgtccatc 8028 catgccgaca cgcacttcgacgacgctctt cgcttcccag cgcggaacac cggggacggg 8088 ctttggcatg tagcatttccctttccagaa gaacgtctgc ccgccgctga tgcgccggta 8148 ttcccgacgc gtgaagatatgctccaaagg cgtttcgggc agcggccggt aggccggttc 8208 agcttcttgc ggcgcgacggcaaactgacg attgtgcttg gcgataagtt ccggtaacac 8268 gcgattggct tcctccatcgtgcacacgtt gcgcagccta agttcgatca ccaggcgatc 8328 ctgaaaggtt tgccagagccgttcgatccg tcctttggct tggggtgaca gcgcctcgat 8388 atgggtaatg cccagatcggcgagggcctg tccgaaggtg gaaagcgacg gcggctcacc 8448 ggccaattcc tgctcgagggttggcttgcc cttgggcggg tgaaaaatgg agtgttggtc 8508 gctgtagagc gcaagcggtacgcctttgcg cctaagtccc tcgatcatga cggtcacgta 8568 gccctccagt gtttcggtcgggcggaaggt ggccgcgacc acttccccgg tggcgtcatc 8628 gatgatgccg tgcagggtgagcatgggacc gcgatcctcc agccaggcat agggagaagc 8688 atcgatctgc cacagcatgcccgcctgagg tttgcggggc cggggtcggt gagccttcgg 8748 acgacggcgc agccgcgcgggacgcaaccc gccttccagc agaatgcggc ggaccgaaga 8808 gacgcttaaa tggatgttttcgtgttcggc caacagctcg gcaaagtggg tggcattgct 8868 tccgaagtag cgctcctgatacaggagcat aacgcgttgt ttgagcgaat cggtcaaggt 8928 gtgagccggc ttacggccccgattcccatg tgcgatcgct tgtgcacctc cgtgacgata 8988 tttggccttg agccgatacgcttgacggac actgatgccc aggttgcgtg caacatcctg 9048 ttccgtgaga tggccgtcgatccatttttc aatgaccata acgcgtttca gttcgttctt 9108 tgtcaaggtg atctgctccttgctcatact gacattttct cggatcagtt acaccctgac 9168 aatatcacag aacaacaacaacaatggctg ggtaatattg acgatttttt ttgcaaatga 9228 tacattaata gtattacaagctgttgtgat tttctttgtc gttattaatt cgacaaagaa 9288 ggggaatgtc ggtacgcttcaaccgacgta taaataatgg gctttattta gccgtggaga 9348 caataggaca cctaatttggtgtctttttg tgtttccgcg gtttttttat gcccaaaaaa 9408 ggaggtaatc gatattggcttcaaatcgtg aagaagtgcg gagcgcggaa cagtatgtgt 9468 tggcggagct gccccaagaattgctcgata ttcgctctta tgatgagtac cacatcaatt 9528 tttcgggcgg ggcagacagcttggccgtag ccattttgat gaaatacggc tataaagtgc 9588 cgccggagaa gcttatcgataccgtcgacc tcgagggggg gcccggtacc cagcttttgt 9648 tccctttagt gagggttaattgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg 9708 tgaaattgtt atccgctcacaattccacac aacatacgag ccgggagcat aaagtgtaaa 9768 gcctggg 9775 6 414 PRTPaenibacillus sp. 6 Met Arg Thr Ile His Ala Asn Ser Ser Ala Val Arg GluAsp His Arg 1 5 10 15 Ala Leu Asp Val Ala Thr Glu Leu Ala Lys Thr PheArg Val Thr Val 20 25 30 Arg Glu Arg Glu Arg Ala Gly Gly Thr Pro Lys AlaGlu Arg Asp Ala 35 40 45 Ile Arg Arg Ser Gly Leu Leu Thr Leu Leu Ile SerLys Glu Arg Gly 50 55 60 Gly Leu Gly Glu Ser Trp Pro Thr Val Tyr Glu AlaIle Ala Glu Ile 65 70 75 80 Ala Ser Ala Asp Ala Ser Leu Gly His Leu PheGly Tyr His Phe Ser 85 90 95 Asn Phe Ala Tyr Val Asp Leu Phe Ala Ser ProGlu Gln Lys Ala Arg 100 105 110 Trp Tyr Pro Gln Ala Val Arg Glu Arg TrpPhe Leu Gly Asn Ala Ser 115 120 125 Ser Glu Asn Asn Ala His Val Leu AspTrp Arg Val Thr Ala Thr Pro 130 135 140 Leu Pro Asp Gly Ser Tyr Glu IleAsn Gly Thr Lys Ala Phe Cys Ser 145 150 155 160 Gly Ser Ala Asp Ala AspArg Leu Leu Val Phe Ala Val Thr Ser Arg 165 170 175 Asp Pro Asn Gly AspGly Arg Ile Val Ala Ala Leu Ile Pro Ser Asp 180 185 190 Arg Ala Gly ValGln Val Asn Gly Asp Trp Asp Ser Leu Gly Met Arg 195 200 205 Gln Thr AspSer Gly Ser Val Thr Phe Ser Gly Val Val Val Tyr Pro 210 215 220 Asp GluLeu Leu Gly Thr Pro Gly Gln Val Thr Asp Ala Phe Ala Ser 225 230 235 240Gly Ser Lys Pro Ser Leu Trp Thr Pro Ile Thr Gln Leu Ile Phe Thr 245 250255 His Leu Tyr Leu Gly Ile Ala Arg Gly Ala Leu Glu Glu Ala Ala His 260265 270 Tyr Ser Arg Ser His Ser Arg Pro Phe Thr Leu Ala Gly Val Glu Lys275 280 285 Ala Thr Glu Asp Pro Tyr Val Leu Ala Ile Tyr Gly Glu Phe AlaAla 290 295 300 Gln Leu Gln Val Ala Glu Ala Gly Ala Arg Glu Val Ala LeuArg Val 305 310 315 320 Gln Glu Leu Trp Glu Arg Asn His Val Thr Pro GluGln Arg Gly Gln 325 330 335 Leu Met Val Gln Val Ala Ser Ala Lys Ile ValAla Thr Arg Leu Val 340 345 350 Ile Glu Leu Thr Ser Arg Leu Tyr Glu AlaMet Gly Ala Arg Ala Ala 355 360 365 Ala Ser Arg Gln Phe Gly Phe Asp ArgPhe Trp Arg Asp Ala Arg Thr 370 375 380 His Thr Leu His Asp Pro Val AlaTyr Lys Ile Arg Glu Val Gly Asn 385 390 395 400 Trp Phe Leu Asn His ArgPhe Pro Thr Pro Ser Phe Tyr Ser 405 410 7 9775 DNA Paenibacillus sp. CDS(641)...(1936) CDS (7026)...(8321) 7 cccaggcttt acactttatg ctcccggctcgtatgttgtg tggaattgtg agcggataac 60 aatttcacac aggaaacagc tatgaccatgattacgccaa gcgcgcaatt aaccctcact 120 aaagggaaca aaagctgggt accgggccccccctcgaggt cgacggtatc gataagcttc 180 tccggcggca ctttatagcc gtatttcatcaaaatggcta cggccaagct gtctgccccg 240 cccgaaaaat tgatgtggta ctcatcataagagcgaatat cgagcaattc ttggggcagc 300 tccgccaaca catactgttc cgcgctccgcacttcttcac gatttgaagc caatatcgat 360 tacctccttt tttgggcata aaaaaaccgcggaaacacaa aaagacacca aattaggtgt 420 cctattgtct ccacggctaa ataaagcccattatttatac gtcggttgaa gcgtaccgac 480 attccccttc tttgtcgaat taataacgacaaagaaaatc acaacagctt gtaatactat 540 taatgtatca tttgcaaaaa aaatcgtcaatattacccag ccattgttgt tgttgttctg 600 tgatattgtc agggtgtaac tgatccgagaaaatgtcagt atg agc aag gag cag 655 Met Ser Lys Glu Gln 1 5 atc acc ttgaca aag aac gaa ctg aaa cgc gtt atg gtc att gaa aaa 703 Ile Thr Leu ThrLys Asn Glu Leu Lys Arg Val Met Val Ile Glu Lys 10 15 20 tgg atc gac ggccat ctc acg gaa cag gat gtt gca cgc aac ctg ggc 751 Trp Ile Asp Gly HisLeu Thr Glu Gln Asp Val Ala Arg Asn Leu Gly 25 30 35 atc agt gtc cgt caagcg tat cgg ctc aag gcc aaa tat cgt cac gga 799 Ile Ser Val Arg Gln AlaTyr Arg Leu Lys Ala Lys Tyr Arg His Gly 40 45 50 ggt gca caa gcg atc gcacat ggg aat cgg ggc cgt aag ccg gct cac 847 Gly Ala Gln Ala Ile Ala HisGly Asn Arg Gly Arg Lys Pro Ala His 55 60 65 acc ttg acc gat tcg ctc aaacaa cgc gtt atg ctc ctg tat cag gag 895 Thr Leu Thr Asp Ser Leu Lys GlnArg Val Met Leu Leu Tyr Gln Glu 70 75 80 85 cgc tac ttc gga agc aat gccacc cac ttt gcc gag ctg ttg gcc gaa 943 Arg Tyr Phe Gly Ser Asn Ala ThrHis Phe Ala Glu Leu Leu Ala Glu 90 95 100 cac gaa aac atc cat tta agcgtc tct tcg gtc cgc cgc att ctg ctg 991 His Glu Asn Ile His Leu Ser ValSer Ser Val Arg Arg Ile Leu Leu 105 110 115 gaa ggc ggg ttg cgt ccc gcgcgg ctg cgc cgt cgt ccg aag gct cac 1039 Glu Gly Gly Leu Arg Pro Ala ArgLeu Arg Arg Arg Pro Lys Ala His 120 125 130 cga ccc cgg ccc cgc aaa cctcag gcg ggc atg ctg tgg cag atc gat 1087 Arg Pro Arg Pro Arg Lys Pro GlnAla Gly Met Leu Trp Gln Ile Asp 135 140 145 gct tct ccc tat gcc tgg ctggag gat cgc ggt ccc atg ctc acc ctg 1135 Ala Ser Pro Tyr Ala Trp Leu GluAsp Arg Gly Pro Met Leu Thr Leu 150 155 160 165 cac ggc atc atc gat gacgcc acc ggg gaa gtg gtc gcg gcc acc ttc 1183 His Gly Ile Ile Asp Asp AlaThr Gly Glu Val Val Ala Ala Thr Phe 170 175 180 cgc ccg acc gaa aca ctggag ggc tac gtg acc gtc atg atc gag gga 1231 Arg Pro Thr Glu Thr Leu GluGly Tyr Val Thr Val Met Ile Glu Gly 185 190 195 ctt agg cgc aaa ggc gtaccg ctt gcg ctc tac agc gac caa cac tcc 1279 Leu Arg Arg Lys Gly Val ProLeu Ala Leu Tyr Ser Asp Gln His Ser 200 205 210 att ttt cac ccg ccc aagggc aag cca acc ctc gag cag gaa ttg gcc 1327 Ile Phe His Pro Pro Lys GlyLys Pro Thr Leu Glu Gln Glu Leu Ala 215 220 225 ggt gag ccg ccg tcg ctttcc acc ttc gga cag gcc ctc gcc gat ctg 1375 Gly Glu Pro Pro Ser Leu SerThr Phe Gly Gln Ala Leu Ala Asp Leu 230 235 240 245 ggc att acc cat atcgag gcg ctg tca ccc caa gcc aaa gga cgg atc 1423 Gly Ile Thr His Ile GluAla Leu Ser Pro Gln Ala Lys Gly Arg Ile 250 255 260 gaa cgg ctc tgg caaacc ttt cag gat cgc ctg gtg atc gaa ctt agg 1471 Glu Arg Leu Trp Gln ThrPhe Gln Asp Arg Leu Val Ile Glu Leu Arg 265 270 275 ctg cgc aac gtg tgcacg atg gag gaa gcc aat cgc gtg tta ccg gaa 1519 Leu Arg Asn Val Cys ThrMet Glu Glu Ala Asn Arg Val Leu Pro Glu 280 285 290 ctt atc gcc aag cacaat cgt cag ttt gcc gtc gcg ccg caa gaa gct 1567 Leu Ile Ala Lys His AsnArg Gln Phe Ala Val Ala Pro Gln Glu Ala 295 300 305 gaa ccg gcc tac cggccg ctg ccc gaa acg cct ttg gag cat atc ttc 1615 Glu Pro Ala Tyr Arg ProLeu Pro Glu Thr Pro Leu Glu His Ile Phe 310 315 320 325 acg cgt cgg gaatac cgg cgc atc agc ggc ggg cag acg ttc ttc tgg 1663 Thr Arg Arg Glu TyrArg Arg Ile Ser Gly Gly Gln Thr Phe Phe Trp 330 335 340 aaa ggg aaa tgctac atg cca aag ccc gtc ccc ggt gtt ccg cgc tgg 1711 Lys Gly Lys Cys TyrMet Pro Lys Pro Val Pro Gly Val Pro Arg Trp 345 350 355 gaa gcg aag agcgtc gtc gaa gtg cgt gtc ggc atg gat gga caa gtg 1759 Glu Ala Lys Ser ValVal Glu Val Arg Val Gly Met Asp Gly Gln Val 360 365 370 tgg ctg tgg gatcaa ggg cgg gcc tgg cct tgt gtg gag aca cag gcc 1807 Trp Leu Trp Asp GlnGly Arg Ala Trp Pro Cys Val Glu Thr Gln Ala 375 380 385 aca cag acc ccggcg cca aca acg gcc aaa aaa gaa gcg gcg cct gcg 1855 Thr Gln Thr Pro AlaPro Thr Thr Ala Lys Lys Glu Ala Ala Pro Ala 390 395 400 405 tcc ccc cgcaag ccc gct gca aac cat ccc tgg aga aaa cca ttc tcc 1903 Ser Pro Arg LysPro Ala Ala Asn His Pro Trp Arg Lys Pro Phe Ser 410 415 420 agc aag cagtta cag cgt agc aca gca tcc ggc tagtctgcaa gaggacggcg 1956 Ser Lys GlnLeu Gln Arg Ser Thr Ala Ser Gly 425 430 gctgtttaat caactatcat ccctgacattttcatagaac agttaacctg acattttcac 2016 agacgcttga cagggggaac tatcatgagtttgagcgagc gaattgacga agcgatccga 2076 aatcatgagg aatgggtgct ccggaggcagaaggggattc ccaataacct gcggctgttt 2136 gtcctggcct gcatggatga acgacttccggtggaagaca ttctcgggct gcagcccggg 2196 gatgcccatg tgttccgcaa tgccggaggggttgtgaccg acgacgtcat ccgctcggcg 2256 gccctcaccc tgaacttctt cgggacgaaagaaatcatcg tgatcaatca caccgaatgc 2316 ggcatgatga ccgccggcgg ccgggaagttgtcgacggtc tccgggccaa actcaacata 2376 gacgtggaac gggttccgtt ggatccctccctccccgagc tggtactgag cgagccggca 2436 gctttcgaaa aatggttccg tacctttgacaatgtggacg aggcgaccgt ggcccaagtc 2496 gaatacctgc gcaaccaccc gctgattccgaaggacacca tcatcaacgg atatatatat 2556 gaagtggaga caaaccgcct gcgccgcccgcaccaattat tgagccgccg ggtgaatacc 2616 cgcgacgaac tgctcgggcg gagataagagaaacgccgat gaaaactccc gtcagggaaa 2676 atgtttttcg ggctgcctca catccaaggggcagcccttc ggtatttatc cggcactaga 2736 gtgaacctgt ccggcccggg gtatgatggaaccgagaggc ttcggtcctg gtccattttc 2796 tcaacgtggt gtttgaagat tccggccatcccctgttgtt cgacgtggaa ccgttgggac 2856 atcgagaagc ggacccttta ctactgggcgaaaatgcttg agagtcacag cacgttccat 2916 ctgcgcgagg atgcgacggg ggtgttgctggccgatctgt tggagataca ttttgtggaa 2976 ctggaatgtc gatctcatcc ggcgttacccccaatcccat cctcaaataa atctattcac 3036 actaaatttc aagagtaaaa gctgggggttggaaaccggt gattgaggaa ccagtttcct 3096 acttcgcgta tcttataggc taccgggtcatgtaaggtat gcgtgcgcgc gtcgcgccaa 3156 aagcggtcaa agccgaattg gcggctcgctgcagcccgtg cccccatcgc ttcatataga 3216 cggcttgtca gttcgatcac caaacgcgtggcgacgattt tggcactggc cacttgtacc 3276 attaactgcc cccgctgctc aggagtgacgtgattccgct cccacaattc ctgaacccgc 3336 aacgccacct ctcgggctcc agcctccgcgacctgaagtt gtgcagcaaa ttccccataa 3396 atcgctagca cataaggatc ctcggtggctttctccaccc ctgcgagtgt aaatggtctc 3456 gaatgggacc tcgagtagtg agcggcctcttcaagagcgc cacgggcaat gccgaggtac 3516 aggtgggtaa agatcagttg ggtgatgggtgtccaaagac tgggcttcga accggaagca 3576 aacgcatccg tcacttggcc gggtgtccccagcaactcgt cgggatagac caccacaccc 3636 gaaaatgtaa cgctcccact atcggtttgacgcataccca ggctgtccca atcgccattt 3696 acctgaaccc cagcacgatc cgaggggatgagtgccgcga cgatcctgcc atctccgttt 3756 ggatccctgc tggtgacggc aaacacaagcaacctgtccg catcggccga gccgctgcaa 3816 aaggccttgg tcccgttgat ctcataactgccgtccggta acggggtcgc cgtcacacgc 3876 caatccagaa cgtgcgcatt gttttcgctggatgcattcc caaggaacca acgctcgcgg 3936 acagcctgtg gataccaacg agccttctgctcaggtgaag caaagagatc cacataggca 3996 aaatttgaaa aatgataacc aaacaggtgcccaagggagg cgtcggcgct ggcaatctca 4056 gcgatggctt cgtatacggt cggccaactttctccgagtc ccccgcgctc tttactgata 4116 agtagagtaa ggaggccact acggcgaatcgcgtcgcgct ccgccttcgg ggttcccccc 4176 gcacgctccc tttcccgaac ggtcacacgaaacgtcttgg ccagttctgt cgccacgtct 4236 aaagcacgat gatcttcacg gactgcagatgaattggcat ggattgttct cattagaaaa 4296 actcctcctc acaaagatgc gttgtccagaaattcggggg ccgcccaacg ctctatatcc 4356 accaaccggt cgatcagccc gtggtcgaaaagaaattgct gggtctgctc cagaatgcgc 4416 agagccgcct gatccagccg cggtcgcaggtccctatgaa acccttcgcc aaatcccgtc 4476 tccacggcct tcacggaaac cccaaggttcaaggcatgga tttcaatggt ctccttggcg 4536 tgatcggttg cccaggacgc agcctccacgacggagtcga ccaagcgttg gacgatctcc 4596 ggcctgcgct ccactagagc cgcgctgaccgtccaaacgc tcgcccaggc attctcctct 4656 cctgtcaacg caaagaccgg cttagccacaccttgaagtt caagctcggc agcataggga 4716 agccaggaaa acagggcgtc tacctgcccgcttttcaact gttgactctg ccattcaacc 4776 gccgtagcaa acaactctcg ggagctgaagtccctactgt catccagttt aggtcgcggg 4836 acatccaccc atgggttttc aacatcttcaacaatgacgt cattcagtct caaaccgccg 4896 gccgccaacg tatgcttgag cgctcgaacttcccacgatc ccagggcgac gagcgtttgc 4956 tcccaagggc caaggttccg atattcctcgcccagatggc cgaaaaggat cctctgtgca 5016 gctcggctca ggccgatgcg gcgccctctaagatccgatg gtgaagcaat cttaccggca 5076 gaatgcacat aaaacccttg gcgaggcttcagaaccgtga ttcccaacaa acgtgtccgc 5136 cccggagcac gcagcccttc gctcaccagcggtggtatct cgccgccaaa tcgggtgtag 5196 gctgcatgat catacgcaaa atgtgtcgccccttgggcaa aggcgatctg cgagagaaga 5256 acaccttgac gctctagctt ccccgtccgtgaggccacga gcaaagcgtt tgcaaccgga 5316 caattactat acgcaacgcg ggtcgggccaatggctgaga gcgtgctcat tcgctcaccc 5376 cctgtacagc ttcttcggct tcgctaacgtcttccagacc gagatgctgg cgcaaggtat 5436 ccccttcata ctcccgtctg aaaagaccccggtcctgcaa aataggaacc actttgtcga 5496 caaattcctc gtaagcgccg ggcagatagtagggagagat cacgaagccg tcggccgccg 5556 gttcattgaa cagagattcc aactcccctgccacctgctc cggggttcca accaactgcg 5616 gtacgagtac ccagccatag cgaacccctaggtcgcgcaa cgttaagttt tctgtctctg 5676 tcagtttcac caccatctct aaaagacctctcgtcccacc cacctcaccg accgcatcca 5736 gcacctcacg aattggagca tccaaaggatagcgggaaaa atccactccg caatggcttg 5796 acagggtaga gagaccagct tctggtatcactagctgatt cacttcttcc tgccgctcgc 5856 gcgcttcctt ctccgtatcg ccaagaatcggaatgacggc agggagtatt ttgcaattct 5916 cgggacggcg tcctgcggcg attacctgttttttcaagtc ttcgtaaaac gcccgcatcg 5976 cgactcggtt cggcgcaatg gtgaaaacggcttctgccca ccgagcagca aatgtctttc 6036 cacgggcgga ggatcccgcc tgaatgatgacaggacgacc ctgtggcgac cgcgggactt 6096 gtaacgggcc ccggacggag aaccaggttccggagtgatc aatatagtgg acctttctgg 6156 gatcagcaaa aagaccctgt tttttgtcgaggagcaatgc atcctgatcc caactcctcc 6216 acaatttatc tgtaatctca agaaactcatcggcacggtc gtaccgacta tcgtgatcca 6276 ggtgttcctc atacccaaag ttcctggcctcggcgttgtt gagtgaggtc acgacattcc 6336 aggctgccct tccttttgtt aagtgatccagcgtagcaaa cactcttgcc acatggtaag 6396 gaggatagta ggttgtcgaa atcgtggcccccaggcccag tctctccgtg gccgcagcca 6456 tggtggcgag cacggggacg ggatccagaaaagcggctcc ttggcctcca aatctcaatc 6516 cagcatcaag attgtttccg tagctatcccaaatagccaa accgtcaggc agaaagagca 6576 ggtcaaagcg gccgcgctcg agtgtacgggcgatgtgttg ataccaagaa atagacaaaa 6636 aaccattatc agttttcggg tgacgccatgccccgtggtg atgggtcaca ttacccgctg 6696 caaaaaaacc ggcaagatgc atttgacgcatcgattgaac acctcccatg tatgttcctg 6756 cgtgaatgtt cttcaatggt catccaggagcagaaaactg tttttcggcc ctcctctgca 6816 aaagaaaaag ccccccgctt tccggcaaaattaccgcaaa gtcagagggc ctctggtggt 6876 ccagtcagcc gaggtaaacc cgattaaattgattagtatt tatgaatcgg agtataccgt 6936 tcaggattca ttttgtcaac ccgtcgcaatcactcatgtt gttgttctgt gatattgtca 6996 gggtgtaact gatccgagaa aatgtcagtatg agc aag gag cag atc acc ttg 7049 Met Ser Lys Glu Gln Ile Thr Leu 435440 aca aag aac gaa ctg aaa cgc gtt atg gtc att gaa aaa tgg atc gac 7097Thr Lys Asn Glu Leu Lys Arg Val Met Val Ile Glu Lys Trp Ile Asp 445 450455 ggc cat ctc acg gaa cag gat gtt gca cgc aac ctg ggc atc agt gtc 7145Gly His Leu Thr Glu Gln Asp Val Ala Arg Asn Leu Gly Ile Ser Val 460 465470 cgt caa gcg tat cgg ctc aag gcc aaa tat cgt cac gga ggt gca caa 7193Arg Gln Ala Tyr Arg Leu Lys Ala Lys Tyr Arg His Gly Gly Ala Gln 475 480485 gcg atc gca cat ggg aat cgg ggc cgt aag ccg gct cac acc ttg acc 7241Ala Ile Ala His Gly Asn Arg Gly Arg Lys Pro Ala His Thr Leu Thr 490 495500 gat tcg ctc aaa caa cgc gtt atg ctc ctg tat cag gag cgc tac ttc 7289Asp Ser Leu Lys Gln Arg Val Met Leu Leu Tyr Gln Glu Arg Tyr Phe 505 510515 520 gga agc aat gcc acc cac ttt gcc gag ctg ttg gcc gaa cac gaa aac7337 Gly Ser Asn Ala Thr His Phe Ala Glu Leu Leu Ala Glu His Glu Asn 525530 535 atc cat tta agc gtc tct tcg gtc cgc cgc att ctg ctg gaa ggc ggg7385 Ile His Leu Ser Val Ser Ser Val Arg Arg Ile Leu Leu Glu Gly Gly 540545 550 ttg cgt ccc gcg cgg ctg cgc cgt cgt ccg aag gct cac cga ccc cgg7433 Leu Arg Pro Ala Arg Leu Arg Arg Arg Pro Lys Ala His Arg Pro Arg 555560 565 ccc cgc aaa cct cag gcg ggc atg ctg tgg cag atc gat gct tct ccc7481 Pro Arg Lys Pro Gln Ala Gly Met Leu Trp Gln Ile Asp Ala Ser Pro 570575 580 tat gcc tgg ctg gag gat cgc ggt ccc atg ctc acc ctg cac ggc atc7529 Tyr Ala Trp Leu Glu Asp Arg Gly Pro Met Leu Thr Leu His Gly Ile 585590 595 600 atc gat gac gcc acc ggg gaa gtg gtc gcg gcc acc ttc cgc ccgacc 7577 Ile Asp Asp Ala Thr Gly Glu Val Val Ala Ala Thr Phe Arg Pro Thr605 610 615 gaa aca ctg gag ggc tac gtg acc gtc atg atc gag gga ctt aggcgc 7625 Glu Thr Leu Glu Gly Tyr Val Thr Val Met Ile Glu Gly Leu Arg Arg620 625 630 aaa ggc gta ccg ctt gcg ctc tac agc gac caa cac tcc att tttcac 7673 Lys Gly Val Pro Leu Ala Leu Tyr Ser Asp Gln His Ser Ile Phe His635 640 645 ccg ccc aag ggc aag cca acc ctc gag cag gaa ttg gcc ggt gagccg 7721 Pro Pro Lys Gly Lys Pro Thr Leu Glu Gln Glu Leu Ala Gly Glu Pro650 655 660 ccg tcg ctt tcc acc ttc gga cag gcc ctc gcc gat ctg ggc attacc 7769 Pro Ser Leu Ser Thr Phe Gly Gln Ala Leu Ala Asp Leu Gly Ile Thr665 670 675 680 cat atc gag gcg ctg tca ccc caa gcc aaa gga cgg atc gaacgg ctc 7817 His Ile Glu Ala Leu Ser Pro Gln Ala Lys Gly Arg Ile Glu ArgLeu 685 690 695 tgg caa acc ttt cag gat cgc ctg gtg atc gaa ctt agg ctgcgc aac 7865 Trp Gln Thr Phe Gln Asp Arg Leu Val Ile Glu Leu Arg Leu ArgAsn 700 705 710 gtg tgc acg atg gag gaa gcc aat cgc gtg tta ccg gaa cttatc gcc 7913 Val Cys Thr Met Glu Glu Ala Asn Arg Val Leu Pro Glu Leu IleAla 715 720 725 aag cac aat cgt cag ttt gcc gtc gcg ccg caa gaa gct gaaccg gcc 7961 Lys His Asn Arg Gln Phe Ala Val Ala Pro Gln Glu Ala Glu ProAla 730 735 740 tac cgg ccg ctg ccc gaa acg cct ttg gag cat atc ttc acgcgt cgg 8009 Tyr Arg Pro Leu Pro Glu Thr Pro Leu Glu His Ile Phe Thr ArgArg 745 750 755 760 gaa tac cgg cgc atc agc ggc ggg cag acg ttc ttc tggaaa ggg aaa 8057 Glu Tyr Arg Arg Ile Ser Gly Gly Gln Thr Phe Phe Trp LysGly Lys 765 770 775 tgc tac atg cca aag ccc gtc ccc ggt gtt ccg cgc tgggaa gcg aag 8105 Cys Tyr Met Pro Lys Pro Val Pro Gly Val Pro Arg Trp GluAla Lys 780 785 790 agc gtc gtc gaa gtg cgt gtc ggc atg gat gga caa gtgtgg ctg tgg 8153 Ser Val Val Glu Val Arg Val Gly Met Asp Gly Gln Val TrpLeu Trp 795 800 805 gat caa ggg cgg gcc tgg cct tgt gtg gag aca cag gccaca cag acc 8201 Asp Gln Gly Arg Ala Trp Pro Cys Val Glu Thr Gln Ala ThrGln Thr 810 815 820 ccg gcg cca aca acg gcc aaa aaa gaa gcg gcg cct gcgtcc ccc cgc 8249 Pro Ala Pro Thr Thr Ala Lys Lys Glu Ala Ala Pro Ala SerPro Arg 825 830 835 840 aag ccc gct gca aac cat ccc tgg aga aaa cca ttctcc agc aag cag 8297 Lys Pro Ala Ala Asn His Pro Trp Arg Lys Pro Phe SerSer Lys Gln 845 850 855 tta cag cgt agc aca gca tcc ggc tagtctgcaagaggacggcg gctgtttaat 8351 Leu Gln Arg Ser Thr Ala Ser Gly 860caactatcat ccctgacatt ttcatagaac agttaacctg acattttcac agacgcttga 8411catattaccc agccattgtc caaccaaaac tctaaaattg tggtatgctg ccgaaatttg 8471aaaagataaa taggaagagt ggaatgctcc acccttccca tttatcacag cgcaaattcc 8531aattgcccgt tcgcggcaag cgacatcggt accacagcag ccgtcatgtc ttgatcttgg 8591 8432 PRT Paenibacillus sp. 8 Met Ser Lys Glu Gln Ile Thr Leu Thr Lys AsnGlu Leu Lys Arg Val 1 5 10 15 Met Val Ile Glu Lys Trp Ile Asp Gly HisLeu Thr Glu Gln Asp Val 20 25 30 Ala Arg Asn Leu Gly Ile Ser Val Arg GlnAla Tyr Arg Leu Lys Ala 35 40 45 Lys Tyr Arg His Gly Gly Ala Gln Ala IleAla His Gly Asn Arg Gly 50 55 60 Arg Lys Pro Ala His Thr Leu Thr Asp SerLeu Lys Gln Arg Val Met 65 70 75 80 Leu Leu Tyr Gln Glu Arg Tyr Phe GlySer Asn Ala Thr His Phe Ala 85 90 95 Glu Leu Leu Ala Glu His Glu Asn IleHis Leu Ser Val Ser Ser Val 100 105 110 Arg Arg Ile Leu Leu Glu Gly GlyLeu Arg Pro Ala Arg Leu Arg Arg 115 120 125 Arg Pro Lys Ala His Arg ProArg Pro Arg Lys Pro Gln Ala Gly Met 130 135 140 Leu Trp Gln Ile Asp AlaSer Pro Tyr Ala Trp Leu Glu Asp Arg Gly 145 150 155 160 Pro Met Leu ThrLeu His Gly Ile Ile Asp Asp Ala Thr Gly Glu Val 165 170 175 Val Ala AlaThr Phe Arg Pro Thr Glu Thr Leu Glu Gly Tyr Val Thr 180 185 190 Val MetIle Glu Gly Leu Arg Arg Lys Gly Val Pro Leu Ala Leu Tyr 195 200 205 SerAsp Gln His Ser Ile Phe His Pro Pro Lys Gly Lys Pro Thr Leu 210 215 220Glu Gln Glu Leu Ala Gly Glu Pro Pro Ser Leu Ser Thr Phe Gly Gln 225 230235 240 Ala Leu Ala Asp Leu Gly Ile Thr His Ile Glu Ala Leu Ser Pro Gln245 250 255 Ala Lys Gly Arg Ile Glu Arg Leu Trp Gln Thr Phe Gln Asp ArgLeu 260 265 270 Val Ile Glu Leu Arg Leu Arg Asn Val Cys Thr Met Glu GluAla Asn 275 280 285 Arg Val Leu Pro Glu Leu Ile Ala Lys His Asn Arg GlnPhe Ala Val 290 295 300 Ala Pro Gln Glu Ala Glu Pro Ala Tyr Arg Pro LeuPro Glu Thr Pro 305 310 315 320 Leu Glu His Ile Phe Thr Arg Arg Glu TyrArg Arg Ile Ser Gly Gly 325 330 335 Gln Thr Phe Phe Trp Lys Gly Lys CysTyr Met Pro Lys Pro Val Pro 340 345 350 Gly Val Pro Arg Trp Glu Ala LysSer Val Val Glu Val Arg Val Gly 355 360 365 Met Asp Gly Gln Val Trp LeuTrp Asp Gln Gly Arg Ala Trp Pro Cys 370 375 380 Val Glu Thr Gln Ala ThrGln Thr Pro Ala Pro Thr Thr Ala Lys Lys 385 390 395 400 Glu Ala Ala ProAla Ser Pro Arg Lys Pro Ala Ala Asn His Pro Trp 405 410 415 Arg Lys ProPhe Ser Ser Lys Gln Leu Gln Arg Ser Thr Ala Ser Gly 420 425 430 9 432PRT Paenibacillus sp. 9 Met Ser Lys Glu Gln Ile Thr Leu Thr Lys Asn GluLeu Lys Arg Val 1 5 10 15 Met Val Ile Glu Lys Trp Ile Asp Gly His LeuThr Glu Gln Asp Val 20 25 30 Ala Arg Asn Leu Gly Ile Ser Val Arg Gln AlaTyr Arg Leu Lys Ala 35 40 45 Lys Tyr Arg His Gly Gly Ala Gln Ala Ile AlaHis Gly Asn Arg Gly 50 55 60 Arg Lys Pro Ala His Thr Leu Thr Asp Ser LeuLys Gln Arg Val Met 65 70 75 80 Leu Leu Tyr Gln Glu Arg Tyr Phe Gly SerAsn Ala Thr His Phe Ala 85 90 95 Glu Leu Leu Ala Glu His Glu Asn Ile HisLeu Ser Val Ser Ser Val 100 105 110 Arg Arg Ile Leu Leu Glu Gly Gly LeuArg Pro Ala Arg Leu Arg Arg 115 120 125 Arg Pro Lys Ala His Arg Pro ArgPro Arg Lys Pro Gln Ala Gly Met 130 135 140 Leu Trp Gln Ile Asp Ala SerPro Tyr Ala Trp Leu Glu Asp Arg Gly 145 150 155 160 Pro Met Leu Thr LeuHis Gly Ile Ile Asp Asp Ala Thr Gly Glu Val 165 170 175 Val Ala Ala ThrPhe Arg Pro Thr Glu Thr Leu Glu Gly Tyr Val Thr 180 185 190 Val Met IleGlu Gly Leu Arg Arg Lys Gly Val Pro Leu Ala Leu Tyr 195 200 205 Ser AspGln His Ser Ile Phe His Pro Pro Lys Gly Lys Pro Thr Leu 210 215 220 GluGln Glu Leu Ala Gly Glu Pro Pro Ser Leu Ser Thr Phe Gly Gln 225 230 235240 Ala Leu Ala Asp Leu Gly Ile Thr His Ile Glu Ala Leu Ser Pro Gln 245250 255 Ala Lys Gly Arg Ile Glu Arg Leu Trp Gln Thr Phe Gln Asp Arg Leu260 265 270 Val Ile Glu Leu Arg Leu Arg Asn Val Cys Thr Met Glu Glu AlaAsn 275 280 285 Arg Val Leu Pro Glu Leu Ile Ala Lys His Asn Arg Gln PheAla Val 290 295 300 Ala Pro Gln Glu Ala Glu Pro Ala Tyr Arg Pro Leu ProGlu Thr Pro 305 310 315 320 Leu Glu His Ile Phe Thr Arg Arg Glu Tyr ArgArg Ile Ser Gly Gly 325 330 335 Gln Thr Phe Phe Trp Lys Gly Lys Cys TyrMet Pro Lys Pro Val Pro 340 345 350 Gly Val Pro Arg Trp Glu Ala Lys SerVal Val Glu Val Arg Val Gly 355 360 365 Met Asp Gly Gln Val Trp Leu TrpAsp Gln Gly Arg Ala Trp Pro Cys 370 375 380 Val Glu Thr Gln Ala Thr GlnThr Pro Ala Pro Thr Thr Ala Lys Lys 385 390 395 400 Glu Ala Ala Pro AlaSer Pro Arg Lys Pro Ala Ala Asn His Pro Trp 405 410 415 Arg Lys Pro PheSer Ser Lys Gln Leu Gln Arg Ser Thr Ala Ser Gly 420 425 430 10 21 PRTPaenibacillus sp VARIANT 2, 5, 17-19 Xaa = any amino acid 10 Met Xaa GlnMet Xaa Leu Ala Gly Phe Phe Ala Ala Gly Asn Val Thr 1 5 10 15 Xaa XaaXaa Gly Ala 20 11 21 PRT Paenibacillus sp. VARIANT 15, 20 Xaa = anyamino acid 11 Thr Lys Ser Ala Ile Gly Pro Thr Arg Val Ala Tyr Ser AsnXaa Pro 1 5 10 15 Val Ala Asn Xaa Leu 20 12 23 PRT Rhodococcus sp 12 MetThr Gln Gln Thr Gln Met His Ala Gly Phe Phe Ser Ala Gly Asn 1 5 10 15Val Thr His Ala His Gly Ala 20 13 23 PRT Rhodococcus sp. 13 Gly Ser GluLeu Asp Ser Ala Ile Arg Asp Thr Leu Thr Tyr Ser Asn 1 5 10 15 Cys ProVal Pro Asn Ala Leu 20 14 26 DNA Artificial Sequence Syntheticallygenerated primer 14 ggnttyttyg cngcnggnaa ygtnac 26 15 17 DNA ArtificialSequence Synthetically generated primer 15 ttygcngcng gnaaygt 17 16 17DNA Artificial Sequence Synthetically generated primer 16 ttyttygcngcnggnaa 17 17 17 DNA Artificial Sequence Synthetically generated primer17 gcnggnttyt tygcngc 17 18 26 DNA Artificial Sequence Syntheticallygenerated primer 18 tangcnacyc tngtnggncc datngc 26 19 17 DNA ArtificialSequence Synthetically generated primer 19 tangcnacyc tngtngg 17 20 17DNA Artificial Sequence Synthetically generated primer 20 tcrttnacngcngtytc 17 21 17 DNA Artificial Sequence Synthetically generated primer21 acyctngtng gnccdat 17

What is claimed is:
 1. An isolated nucleic acid comprising a sequencethat encodes: a protein comprising an amino acid sequence of SEQ ID NO:4.
 2. A vector comprising: a sequence that encodes a polypeptide with anamino acid sequence of SEQ ID NO:
 4. 3. A transformed cell comprising: avector comprising a sequence that encodes a polypeptide with an aminoacid sequence of SEQ ID NO:
 4. 4. A transformed cell comprising: a firstvector comprising a sequence that encodes a first polypeptide forconverting a first compound into a second compound; a second vectorcomprising a sequence that encodes a second polypeptide for convertingthe second compound into a third compound; and a third vector comprisinga sequence that encodes a third polypeptide with an amino acid sequenceof SEQ ID NO: 4 for converting the third compound into a fourthcompound.
 5. The transformed cell according to claim 4 wherein thefirst, second, and third compounds are sulfur compounds; and the fourthcompound is a desulfurized compound.
 6. The transformed cell accordingto claim 4 wherein the first compound comprises dibenzothiophene; thesecond compound comprises dibenzothiophenesulfone; the third compoundcomprises 2-(2′-hydroxyphenyl) benzenesulfinic acid; and the fourthcompound comprises 2-hydroxylbiphenyl.
 7. A transformed cell accordingto claim 4 wherein the first polypeptide comprises an amino acidsequence of SEQ ID NO: 6; and the second polypeptide comprises an aminoacid sequence of SEQ ID NO:
 2. 8. The transformed cell according toclaim 4, wherein the second polypeptide has the followingcharacteristics: (1) Function: it converts dibenzothiophenesulfone into2-(2′-hydroxyphenyl) benzenesulfinic acid (2) Optimum pH: 5.5, stablepH: 5-10; (3) Optimum temperature: 45° C. (4) Molecular weight: 120,000(as determined by gel filtration); (5) Inhibition of activity: it isinhibited by chelating agents or SH inhibitors, but no by 2-HBP orsulfate; and (6) Requirement for coenzyme: NADH and FMN are required;NADPH can be substituted for NADH, but FAD cannot be substituted forFMN.
 9. The transformed cell according to claim 4, wherein the thirdpolypeptide has the following characteristics: (1) Function:2-(2′-hydroxyphenyl) benzenesulfinic acid into 2-hydroxylbiphenyl; (2)Optimum pH: 8, stable pH: 5.5-9.5; (3) Optimum temperature: 55° C.; (4)Molecular weight: 31,000 (as determined by gel filtration); (5)Inhibition of activity: it is inhibited by chelating agents and SHinhibitors, but not by 2-HBP or sulfate; and (6) Requirement forcoenzyme; coenzyme is not required.
 10. A process of producingpolypeptides comprising: expressing in the transformed cell of claim 4,the first polypeptide to convert the first compound into the secondcompound; expressing in the transformed cell the second polypeptide toconvert the second compound into the third compound; and expressing inthe transformed cell the third polypeptide to convert the third compoundinto the fourth compound.
 11. A process of producing polypeptidescomprising: expressing in the transformed cell of claim 4, the firstpolypeptide to convert dibenzothiophene into dibenzothiophenesulfone;expressing in the transformed cell the second polypeptide to convertdibenzothiophenesulfone into 2-(2′-hydroxyphenyl) benzenesulfinic acid;and expressing in the transformed cell the third polypeptide to convert2-(2′-hydroxyphenyl) benzenesulfinic acid into 2-hydroxylbiphenyl.
 12. Aprocess of producing a transformed cell comprising: providing into acell a first vector comprising a sequence that encodes a firstpolypeptide to convert a first compound into a second compound;providing into the cell a second vector comprising a sequence thatencodes a second polypeptide to convert the second compound into a thirdcompound; and providing into the cell a third vector comprising asequence that encodes a third polypeptide with an amino acid sequence ofSEQ ID NO: 4 to convert the third compound into a fourth compound. 13.The process according to claim 12 wherein the first compound comprisesdibenzothiophene; the second compound comprises dibenzothiophenesulfonethe third compound comprises 2-(2′-hydroxyphenyl) benzenesulfinic acid;and the fourth compound comprises 2-hydroxylbiphenyl.
 14. A process ofproducing a transformed cell according to claim 12 wherein: the firstpolypeptide comprises an amino acid sequence of SEQ ID NO: 6; and thesecond polypeptide comprises an amino acid sequence of SEQ ID NO:
 2. 15.The process according to claim 12, wherein the second polypeptide hasthe following characteristics: (1) Function: it convertsdibenzothiophenesulfone into 2-(2′-hydroxyphenyl) benzenesulfinic acid(2) Optimum pH: 5.5, stable pH: 5-10; (3) Optimum temperature: 45° C.(4) Molecular weight: 120,000 (as determined by gel filtration); (5)Inhibition of activity: it is inhibited by chelating agents or SHinhibitors, but no by 2-HBP or sulfate; and (6) Requirement forcoenzyme: NADH and FMN are required; NADPH can be substituted for NADH,but FAD cannot be substituted for FMN.
 16. The process according toclaim 12, wherein the third polypeptide has the followingcharacteristics: (1) Function: 2-(2′-hydroxyphenyl) benzenesulfinic acidinto 2-hydroxylbiphenyl (2) Optimum pH: 8, stable pH: 5.5-9.5; (3)Optimum temperature: 55° C.; (4) Molecular weight: 31,000 (as determinedby gel filtration); (5) Inhibition of activity: it is inhibited bychelating agents and SH inhibitors, but not by 2-HBP or sulfate; and (6)Requirement for coenzyme; coenzyme is not required.
 17. The processaccording to claim 12 wherein the first, second, and third compounds aresulfur compounds; and the fourth compound is a desulfurized compound.